Modified cry3a toxins and nucleic acid sequences coding therefor

ABSTRACT

Methods for making a modified Cry3A toxin are disclosed. Such methods include the insertion of a protease recognition site that is recognized by a gut protease of a target insect, such as corn rootworm, into at least one position of a Cry3A toxin so that a modified Cry3A toxin is thus designed. The coding sequence of the modified Cry3A toxin may be transformed into a host cell and the host cell grown under conditions that allow the host cell to produce the modified Cry3A toxin. The host cell may be a plant cell and the plant may be comprised in a transgenic plant. Thus, the transgenic plant may be used to produce the modified Cry3A toxin.

This application is a continuation of co-pending U.S. patent applicationSer. No. 12/488,759, filed Jun. 22, 2009, which is a continuation ofU.S. patent application Ser. No. 11/834,037, filed Aug. 6, 2007, nowU.S. Pat. No. 7,569,363, which is a divisional of U.S. patentapplication Ser. No. 11/294,220, filed Dec. 5, 2005, now U.S. Pat. No.7,276,583, which is a divisional of U.S. patent application Ser. No.10/229,346, filed Aug. 27, 2002, now U.S. Pat. No. 7,030,295, whichclaims the benefit of U.S. Provisional Application No. 60/316,421, filedAug. 31, 2001, all of which are incorporated herein by reference intheir entirety.

FIELD OF THE INVENTION

The present invention relates to the fields of protein engineering,plant molecular biology and pest control. More particularly, the presentinvention relates to novel modified Cry3A toxins and nucleic acidsequences whose expression results in the modified Cry3A toxins, andmethods of making and methods of using the modified Cry3A toxins andcorresponding nucleic acid sequences to control insects.

BACKGROUND OF THE INVENTION

Species of corn rootworm are considered to be the most destructive cornpests. In the United States the three important species are Diabroticavirgifera virgifera, the western corn rootworm; D. longicornis barberi,the northern corn rootworm and D. undecimpunctata howardi, the southerncorn rootworm. Only western and northern corn rootworms are consideredprimary pests of corn in the US Corn Belt. Corn rootworm larvae causethe most substantial plant damage by feeding almost exclusively on cornroots. This injury has been shown to increase plant lodging, to reducegrain yield and vegetative yield as well as alter the nutrient contentof the grain. Larval feeding also causes indirect effects on maize byopening avenues through the roots for bacterial and fungal infectionswhich lead to root and stalk rot diseases. Adult corn rootworms areactive in cornfields in late summer where they feed on ears, silks andpollen, interfering with normal pollination.

Corn rootworms are mainly controlled by intensive applications ofchemical pesticides, which are active through inhibition of insectgrowth, prevention of insect feeding or reproduction, or cause death.Good corn rootworm control can thus be reached, but these chemicals cansometimes also affect other, beneficial organisms. Another problemresulting from the wide use of chemical pesticides is the appearance ofresistant insect varieties. Yet another problem is due to the fact thatcorn rootworm larvae feed underground thus making it difficult to applyrescue treatments of insecticides. Therefore, most insecticideapplications are made prophylactically at the time of planting. Thispractice results in a large environmental burden. This has beenpartially alleviated by various farm management practices, but there isan increasing need for alternative pest control mechanisms.

Biological pest control agents, such as Bacillus thuringiensis (Bt)strains expressing pesticidal toxins like δ-endotoxins, have also beenapplied to crop plants with satisfactory results against primarilylepidopteran insect pests. The δ-endotoxins are proteins held within acrystalline matrix that are known to possess insecticidal activity wheningested by certain insects. The various δ-endotoxins have beenclassified based upon their spectrum of activity and sequence homology.Prior to 1990, the major classes were defined by their spectrum ofactivity with the Cry1 proteins active against Lepidoptera (moths andbutterflies), Cry2 proteins active against both Lepidoptera and Diptera(flies and mosquitoes), Cry3 proteins active against Coleoptera(beetles) and Cry4 proteins active against Diptera (Hofte and Whitely,1989, Microbiol. Rev. 53:242-255). Recently a new nomenclature wasdeveloped which systematically classifies the Cry proteins based onamino acid sequence homology rather than insect target specificities(Crickmore et al. 1998, Microbiol. Molec. Biol. Rev. 62:807-813).

The spectrum of insecticidal activity of an individual δ-endotoxin fromBt is quite narrow, with a given δ-endotoxin being active against only afew species within an Order. For instance, the Cry3A protein is known tobe very toxic to the Colorado potato beetle, Leptinotarsa decemlineata,but has very little or no toxicity to related beetles in the genusDiabrotica (Johnson et al., 1993, J. Econ. Entomol. 86:330-333).According to Slaney et al. (1992, Insect Biochem. Molec. Biol. 22:9-18)the Cry3A protein is at least 2000 times less toxic to southern cornrootworm larvae than to the Colorado potato beetle. It is also knownthat Cry3A has little or no toxicity to the western corn rootworm.

Specificity of the δ-endotoxins is the result of the efficiency of thevarious steps involved in producing an active toxin protein and itssubsequent interaction with the epithelial cells in the insect mid-gut.To be insecticidal, most known δ-endotoxins must first be ingested bythe insect and proteolytically activated to form an active toxin.Activation of the insecticidal crystal proteins is a multi-step process.After ingestion, the crystals must first be solubilized in the insectgut. Once solubilized, the δ-endotoxins are activated by specificproteolytic cleavages. The proteases in the insect gut can play a rolein specificity by determining where the δ-endotoxin is processed. Oncethe δ-endotoxin has been solubilized and processed it binds to specificreceptors on the surface of the insects' mid-gut epithelium andsubsequently integrates into the lipid bilayer of the brush bordermembrane. Ion channels then form disrupting the normal function of themidgut eventually leading to the death of the insect.

In Lepidoptera, gut proteases process δ-endotoxins from 130-140 kDaprotoxins to toxic proteins of approximately 60-70 kDa. Processing ofthe protoxin to toxin has been reported to proceed by removal of both N-and C-terminal amino acids with the exact location of processing beingdependent on the specific insect gut fluids involved (Ogiwara et al.,1992, J. Invert. Pathol. 60:121-126). The proteolytic activation of a8-endotoxin can play a significant role in determining its specificity.For example, a δ-endotoxin from Bt var. aizawa, called IC1, has beenclassified as a Cry1Ab protein based on its sequence homology with otherknown Cry1 Ab proteins. Cry1Ab proteins are typically active againstlepidopteran insects. However, the IC1 protein has activity against bothlepidopteran and dipteran insects depending upon how the protein isprocessed (Haider et al. 1986, Euro. J. Biochem. 156: 531-540). In adipteran gut, a 53 kDa active IC1 toxin is obtained, whereas in alepidopteran gut, a 55 kDa active IC1 toxin is obtained. IC1 differsfrom the holotype HD-1 Cry1Ab protein by only four amino acids, so grosschanges in the receptor binding region do not seem to account for thedifferences in activity. The different proteolytic cleavages in the twodifferent insect guts possibly allow the activated molecules to folddifferently thus exposing different regions capable of binding differentreceptors. The specificity therefore, appears to reside with the gutproteases of the different insects.

Coleopteran insects have guts that are more neutral to acidic andcoleopteran-specific δ-endotoxins are similar to the size of theactivated lepidopteran-specific toxins. Therefore, the processing ofcoleopteran-specific δ-endotoxins was formerly considered unnecessaryfor toxicity. However, recent data suggests that coleopteran-activeδ-endotoxins are solubilized and proteolyzed to smaller toxicpolypeptides. The 73 kDa Cry3A δ-endotoxin protein produced by B.thuringiensis var. tenebrionis is readily processed in the bacterium atthe N-terminus, losing 49-57 residues during or after crystal formationto produce the commonly isolated 67 kDa form (Carroll et al., 1989,Biochem. J. 261:99-105). McPherson et al., 1988 (Biotechnology 6:61-66)also demonstrated that the native cry3A gene contains two functionaltranslational initiation codons in the same reading frame, one codingfor the 73 kDa protein and the other coding for the 67 kDa proteinstarting at Met-1 and Met-48 respectively, of the deduced amino acidsequence (See SEQ ID NO: 2). Both proteins then can be considerednaturally occurring full-length Cry3A proteins. Treatment of soluble 67kDa Cry3A protein with either trypsin or insect gut extract results in acleavage product of 55 kDa with Asn-159 of the deduced amino acidsequence at the N-terminus. This polypeptide was found to be as toxic toa susceptible coleopteran insect as the native 67 kDa Cry3A toxin.(Carroll et al. Ibid). Thus, a natural trypsin recognition site existsbetween Arg-158 and Asn-159 of the deduced amino acid sequence of thenative Cry3A toxin (SEQ ID NO: 2). Cry3A can also be cleaved bychymotrypsin, resulting in three polypeptides of 49, 11, and 6 kDa.N-terminal analysis of the 49 and 6 kDa components showed the firstamino acid residue to be Ser-162 and Tyr-588, respectively (Carroll etal., 1997 J. Invert. Biol. 70:41-49). Thus, natural chymotrypsinrecognition sites exist in Cry3A between His-161 and Ser-162 and betweenTyr-587 and Tyr-588 of the deduced amino acid sequence (SEQ ID NO: 2).The 49 kDa chymotrypsin product appears to be more soluble at neutral pHthan the native 67 kDa protein or the 55 kDa trypsin product and retainsfull insecticidal activity against the Cry3A-susceptible insects,Colorado potato beetle and mustard beetle, (Phaedon cochleariae).

Insect gut proteases typically function in aiding the insect inobtaining needed amino acids from dietary protein. The best understoodinsect digestive proteases are serine proteases that appear to be themost common (Englemann and Geraerts, 1980, J. Insect Physiol.261:703-710), particularly in lepidopteran species. The majority ofcoleopteran larvae and adults, for example Colorado potato beetle, haveslightly acidic midguts, and cysteine proteases provide the majorproteolytic activity (Wolfson and Mudock, 1990, J. Chem. Ecol.16:1089-1102). More precisely, Thie and Houseman (1990, Insect Biochem,20:313-318) identified and characterized the cysteine proteases,cathepsin B and 1-1, and the aspartyl protease, cathepsin D in Coloradopotato beetle. Gillikin et al. (1992, Arch. Insect Biochem. Physiol.19:285-298) characterized the proteolytic activity in the guts ofwestern corn rootworm larvae and found 15, primarily cysteine,proteases. Until disclosed in this invention, no reports have indicatedthat the serine protease, cathepsin G, exists in western corn rootworm.The diversity and different activity levels of the insect gut proteasesmay influence an insect's sensitivity to a particular Bt toxin.

Many new and novel Bt strains and δ-endotoxins with improved or novelbiological activities have been described over the past five yearsincluding strains active against nematodes (EP 0517367A1). However,relatively few of these strains and toxins have activity againstcoleopteran insects. Further, none of the now known coleopteran-active8-endotoxins, for example Cry3A, Cry3B, Cry3C, Cry7A, Cry8A, Cry8B, andCry8C, have sufficient oral toxicity against corn rootworm to provideadequate field control if delivered, for example, through microbes ortransgenic plants. Therefore, other approaches for producing noveltoxins active against corn rootworm need to be explored.

As more knowledge has been gained as to how the 8-endotoxins function,attempts to engineer 8-endotoxins to have new activities have increased.Engineering 8-endotoxins was made more possible by the solving of thethree dimensional structure of Cry3A in 1991 (Li et al., 1991, Nature353:815-821). The protein has three structural domains: the N-terminaldomain I, from residues 1-290, consists of 7 alpha helices, domain II,from residues 291-500, contains three beta-sheets and the C-terminaldomain III, from residues 501-644, is a beta-sandwich. Based on thisstructure, a hypothesis has been formulated regarding thestructure/function relationship of the δ-endotoxins. It is generallythought that domain I is primarily responsible for pore formation in theinsect gut membrane (Gazit and Shai, 1993, Appl. Environ. Microbiol.57:2816-2820), domain II is primarily responsible for interaction withthe gut receptor (Ge et al., 1991, J. Biol. Chem. 32:3429-3436) and thatdomain III is most likely involved with protein stability (Li et al.1991, supra) as well as having a regulatory impact on ion channelactivity (Chen et al., 1993, PNAS 90:9041-9045).

Lepidopteran-active δ-endotoxins have been engineered in attempts toimprove specific activity or to broaden the spectrum of insecticidalactivity. For example, the silk moth (Bombyx mori) specificity domainfrom Cry1Aa was moved to Cry1Ac, thus imparting a new insecticidalactivity to the resulting chimeric protein (Ge et al. 1989, PNAS 86:4037-4041). Also, Bosch et al. 1998 (U.S. Pat. No. 5,736,131), created anew lepidopteran-active toxin by substituting domain III of Cry1E withdomain III of Cry1C thus producing a Cry1E-Cry1C hybrid toxin with abroader spectrum of lepidopteran activity.

Several attempts at engineering the coleopteran-active δ-endotoxins havebeen reported. Van Rie et al., 1997, (U.S. Pat. No. 5,659,123)engineered Cry3A by randomly replacing amino acids, thought to beimportant in solvent accessibility, in domain II with the amino acidalanine. Several of these random replacements confined to receptorbinding domain II were reportedly involved in increased western cornrootworm toxicity. However, others have shown that some alaninereplacements in domain II of Cry3A result in disruption of receptorbinding or structural instability (Wu and Dean, 1996, J. Mol. Biol. 255:628-640). English et al., 1999, (Intl. Pat. Appl. Publ. No. WO 99/31248)reported amino acid substitutions in Cry3Bb that caused increases intoxicity to southern and western corn rootworm. However, of the 35reported Cry3Bb mutants, only three, with mutations primarily in domainII and the domain II-domain I interface, were active against westerncorn rootworm. Further, the differences in toxicity of wild-type Cry3Bbagainst western corn rootworm in the same assays were greater than anyof the differences between the mutated Cry3Bb toxins and the wild-typeCry3Bb. Therefore, improvements in toxicity of the Cry3Bb mutants appearto be confined primarily to southern corn rootworm.

There remains a need to design new and effective pest control agentsthat provide an economic benefit to farmers and that are environmentallyacceptable. Particularly needed are modified Cry3A toxins that controlwestern corn rootworm, the major pest of corn in the United States, thatare or could become resistant to existing insect control agents.Furthermore, agents whose application minimizes the burden on theenvironment, as through transgenic plants, are desirable.

SUMMARY

In view of these needs, it is an object of the present invention toprovide novel nucleic acid sequences encoding modified Cry3A toxinshaving increased toxicity to corn rootworm. By inserting a proteaserecognition site that is recognized by a target-insect gut protease inat least one position of a Cry3A toxin, in accordance with the presentinvention, a modified Cry3A toxin having significantly greater toxicity,particularly to western and northern corn rootworm is designed. Theinvention is further drawn to the novel modified Cry3A toxins resultingfrom the expression of the nucleic acid sequences, and to compositionsand formulations containing the modified Cry3A toxins, which are capableof inhibiting the ability of insect pests to survive, grow andreproduce, or of limiting insect-related damage or loss to crop plants.The invention is further drawn to a method of making the modified Cry3Atoxins and to methods of using the modified cry3A nucleic acidsequences, for example in microorganisms to control insects or intransgenic plants to confer protection from insect damage, and to amethod of using the modified Cry3A toxins, and compositions andformulations comprising the modified Cry3A toxins, for example applyingthe modified Cry3A toxins or compositions or formulations toinsect-infested areas, or to prophylactically treat insect-susceptibleareas or plants to confer protection against the insect pests.

The novel modified Cry3A toxins described herein are highly activeagainst insects. For example, the modified Cry3A toxins of the presentinvention can be used to control economically important insect pestssuch as western corn rootworm (Diabrotica virgifera virgifera) andnorthern corn rootworm (D. longicornis barberi). The modified Cry3Atoxins can be used singly or in combination with other insect controlstrategies to confer maximal pest control efficiency with minimalenvironmental impact.

According to one aspect, the present invention provides an isolatednucleic acid molecule comprising a nucleotide sequence that encodes amodified Cry3A toxin, wherein the modified Cry3A toxin comprises atleast one additional protease recognition site that does not naturallyoccur in a Cry3A toxin. The additional protease recognition site, whichis recognized by a gut protease of a target insect, is inserted atapproximately the same position as a naturally occurring proteaserecognition site in the Cry3A toxin. The modified Cry3A toxin causeshigher mortality to a target insect than the mortality caused by a Cry3Atoxin to the same target insect. Preferably, the modified Cry3A toxincauses at least about 50% mortality to a target insect to which a Cry3Atoxin causes only up to about 30% mortality.

In one embodiment of this aspect, the gut protease of a target insect isselected from the group consisting of serine proteases, cysteineproteases and aspartic proteases. Preferable serine proteases accordingto this embodiment include cathepsin G, trypsin, chymotrypsin,carboxypeptidase, endopeptidase and elastase, most preferably cathepsinG.

In another embodiment of this aspect, the additional proteaserecognition site is inserted in either domain I or domain III or in bothdomain I and domain III of the Cry3A toxin. Preferably, the additionalprotease recognition site is inserted in either domain I or domain IIIor in both domain I and domain III at a position that replaces, isadjacent to, or is within a naturally occurring protease recognitionsite.

In a yet another embodiment, the additional protease recognition site isinserted in domain I between amino acids corresponding to amino acidnumbers 154 and 162 of SEQ ID NO: 2. Preferably, the additional proteaserecognition site is inserted between amino acid numbers 154 and 162 ofSEQ ID NO: 2 or between amino acid numbers 107 and 115 of SEQ ID NO: 4.

In still another embodiment, the additional protease recognition site isinserted between amino acids corresponding to amino acid numbers 154 and160 of SEQ ID NO: 2. Preferably, the additional protease recognitionsite is inserted between amino acid numbers 154 and 160 of SEQ ID NO: 2or between amino acid numbers 107 and 113 of SEQ ID NO: 4.

In a further embodiment, the additional protease recognition site isinserted in domain I between amino acids corresponding to amino acidnumbers 154 and 158 of SEQ ID NO: 2. Preferably, the additional proteaserecognition site is inserted in domain I between amino acid numbers 154and 158 of SEQ ID NO: 2 or between amino acid numbers 107 and 111 of SEQID NO: 4.

In another embodiment, the additional protease recognition site isinserted in domain III between amino acids corresponding to amino acidnumbers 583 and 589 of SEQ ID NO: 2. Preferably, the additional proteasesite is inserted in domain III between amino acid numbers 583 and 589 ofSEQ ID NO: 2 or between amino acid numbers 536 and 542 of SEQ ID NO: 4.

In still another embodiment, the additional protease recognition site isinserted in domain III between amino acids corresponding to amino acidnumbers 583 and 588 of SEQ ID NO: 2. Preferably, the additional proteasesite is inserted in domain III between amino acid numbers 583 and 588 ofSEQ ID NO: 2 or between amino acid numbers 536 and 541 of SEQ ID NO: 4.

In yet another embodiment, the additional protease recognition site isinserted in domain III between amino acids corresponding to amino acidnumbers 587 and 588 of SEQ ID NO: 2. Preferably, the additional proteasesite is inserted in domain III between amino acid numbers 587 and 588 ofSEQ ID NO: 2 or between amino acid numbers 540 and 541 of SEQ ID NO: 4.

In one embodiment, the additional protease recognition site is insertedin domain I and domain III of the unmodified Cry3A toxin. Preferably,the additional protease recognition site is inserted in domain I at aposition that replaces or is adjacent to a naturally occurring proteaserecognition site and in domain 111 at a position that is within,replaces, or is adjacent to a naturally occurring protease recognitionsite.

In another embodiment, the additional protease recognition site isinserted in domain I between amino acids corresponding to amino acidnumbers 154 and 160 and in domain III between amino acids correspondingto amino acid numbers 587 and 588 of SEQ ID NO: 2. Preferably, theadditional protease recognition site is inserted in domain I betweenamino acid numbers 154 and 160 and in domain III between amino acidnumbers 587 and 588 of SEQ ID NO: 2 or in domain I between amino acidnumbers 107 and 113 and in domain III between amino acid numbers 540 and541 of SEQ ID NO: 4.

In yet another embodiment, the additional protease recognition site islocated in domain I between amino acids corresponding to amino acidnumbers 154 and 158 and in domain III between amino acids correspondingto amino acid numbers 587 and 588 of SEQ ID NO: 2. Preferably, theadditional protease recognition site is inserted in domain I betweenamino acid numbers 154 and 158 and in domain III between amino acidnumbers 587 and 588 of SEQ ID NO: 2 or in domain I between amino acidnumbers 107 and 111 and in domain III between amino acid numbers 540 and541 of SEQ ID NO: 4.

In another embodiment, the additional protease recognition site islocated in domain I between amino acids corresponding to amino acidnumbers 154 and 158 and in domain III between amino acids correspondingto amino acid numbers 583 and 588 of SEQ ID NO: 2. Preferably, theadditional protease recognition site is inserted in domain I betweenamino acid numbers 154 and 158 and in domain III between amino acidnumbers 583 and 588 of SEQ ID NO: 2 or in domain I between amino acidnumbers 107 and 111 and in domain III between amino acid numbers 536 and541 of SEQ ID NO: 4.

In a preferred embodiment, the isolated nucleic acid molecule of thepresent invention comprises nucleotides 1-1791 of SEQ ID NO: 6,nucleotides 1-1806 of SEQ ID NO: 8, nucleotides 1-1818 of SEQ ID NO: 10,nucleotides 1-1794 of SEQ ID NO: 12, nucleotides 1-1812 of SEQ ID NO:14, nucleotides 1-1812 of SEQ ID NO: 16, nucleotides 1-1818 of SEQ IDNO: 18, or nucleotides 1-1791 of SEQ ID NO: 20.

In another preferred embodiment, the isolated nucleic acid molecule ofthe invention encodes a modified Cry3A toxin comprising the amino acidsequence set forth in SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ IDNO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, or SEQ ID NO: 21.

According to one embodiment of the invention, the isolated nucleic acidmolecule encodes a modified Cry3A toxin that is active against acoleopteran insect. Preferably, the modified Cry3A toxin has activityagainst western corn rootworm.

The present invention provides a chimeric gene comprising a heterologouspromoter sequence operatively linked to the nucleic acid molecule of theinvention. The present invention also provides a recombinant vectorcomprising such a chimeric gene. Further, the present invention providesa transgenic non-human host cell comprising such a chimeric gene. Atransgenic host cell according to this aspect of the invention may be abacterial cell or a plant cell, preferably, a plant cell. The presentinvention further provides a transgenic plant comprising such a plantcell. A transgenic plant according to this aspect of the invention maybe sorghum, wheat, sunflower, tomato, potato, cole crops, cotton, rice,soybean, sugar beet, sugarcane, tobacco, barley, oilseed rape, or maize,preferably, maize. The present invention also provides seed from thegroup of transgenic plants consisting of sorghum, wheat, sunflower,tomato, potato, cole crops, cotton, rice, soybean, sugar beet,sugarcane, tobacco, barley, oilseed rape, and maize. In a particularlypreferred embodiment, the seed is from a transgenic maize plant.

In another aspect, the present invention provides toxins produced by theexpression of the nucleic acid molecules of the present invention. In apreferred embodiment, the toxin is produced by the expression of thenucleic acid molecule comprising nucleotides 1-1791 of SEQ ID NO: 6,nucleotides 1-1806 of SEQ ID NO: 8, nucleotides 1-1818 of SEQ ID NO: 10,nucleotides 1-1794 of SEQ ID NO: 12, nucleotides 1-1812 of SEQ ID NO:14, nucleotides 1-1812 of SEQ ID NO: 16, nucleotides 1-1818 of SEQ IDNO: 18, or nucleotides 1-1791 of SEQ ID NO: 20.

In another embodiment, the toxins of the invention are active againstcoleopteran insects, preferably against western corn rootworm.

In one embodiment, a toxin of the present invention comprises the aminoacid sequence set forth in SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11,SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, or SEQ IDNO: 21.

The present invention also provides a composition comprising aneffective insect-controlling amount of a toxin according to theinvention.

In another aspect, the present invention provides a method of producinga toxin that is active against insects, comprising: (a) obtaining a hostcell comprising a chimeric gene, which itself comprises a heterologouspromoter sequence operatively linked to the nucleic acid molecule of theinvention; and (b) expressing the nucleic acid molecule in thetransgenic host cell, which results in at least one toxin that is activeagainst insects.

In a further aspect, the present invention provides a method ofproducing an insect-resistant transgenic plant, comprising introducing anucleic acid molecule of the invention into the transgenic plant,wherein the nucleic acid molecule is expressible in the transgenic plantin an effective amount to control insects. In a preferred embodiment,the insects are coleopteran insects, preferably western corn rootworm.

In yet a further aspect, the present invention provides a method ofcontrolling insects, comprising delivering to the insects an effectiveamount of a toxin of the invention. According to one embodiment, theinsects are coleopteran insects, preferably, western corn rootworm.

Preferably, the toxin is delivered to the insects orally. In onepreferred embodiment, the toxin is delivered orally through a transgenicplant comprising a nucleic acid sequence that expresses a toxin of thepresent invention.

Also provided by the present invention is a method of making a modifiedCry3A toxin, comprising: (a) obtaining a cry3A toxin gene which encodesa Cry3A toxin; (b) identifying a gut protease of a target insect; (c)obtaining a nucleotide sequence which encodes a recognition sequence forthe gut protease; (d) inserting the nucleotide sequence of (c) intoeither domain I or domain III or both domain I and domain III at aposition that replaces, is within, or adjacent to a nucleotide sequencethat codes for a naturally occurring protease recognition site in acry3A toxin gene, thus creating a modified cry3A toxin gene; (e)inserting the modified cry3A toxin gene in an expression cassette; (f)expressing the modified cry3A toxin gene in a non-human host cell,resulting in the host cell producing a modified Cry3A toxin; and, (g)bioassaying the modified Cry3A toxin against a target insect, wherebythe modified Cry3A toxin causes higher mortality to the target insectthan the mortality caused by a Cry3A toxin. In a preferred embodiment,the modified Cry3A toxin causes at least about 50% mortality to thetarget insect when the Cry3A toxin causes up to about 30% mortality.

The present invention further provides a method of controlling insectswherein the transgenic plant further comprises a second nucleic acidsequence or groups of nucleic acid sequences that encode a secondpesticidal principle. Particularly preferred second nucleic acidsequences are those that encode a δ-endotoxin, those that encode aVegetative Insecticidal Protein toxin, disclosed in U.S. Pat. Nos.5,849,870 and 5,877,012, incorporated herein by reference, or those thatencode a pathway for the production of a non-proteinaceous pesticidalprinciple.

Yet another aspect of the present invention is the provision of a methodfor mutagenizing a nucleic acid molecule according to the presentinvention, wherein the nucleic acid molecule has been cleaved intopopulations of double-stranded random fragments of a desired size,comprising: (a) adding to the population of double-stranded randomfragments one or more single- or double-stranded oligonucleotides,wherein the oligonucleotides each comprise an area of identity and anarea of heterology to a double-stranded template polynucleotide; (b)denaturing the resultant mixture of double-stranded random fragments andoligonucleotides into single-stranded fragments; (c) incubating theresultant population of single-stranded fragments with polymerase underconditions which result in the annealing of the single-strandedfragments at the areas of identity to form pairs of annealed fragments,the areas of identity being sufficient for one member of the pair toprime replication of the other, thereby forming a mutagenizeddouble-stranded polynucleotide; and (d) repeating the second and thirdsteps for at least two further cycles, wherein the resultant mixture inthe second step of a further cycle includes the mutagenizeddouble-stranded polynucleotide from the third step of the previouscycle, and wherein the further cycle forms a further mutagenizeddouble-stranded polynucleotide.

Other aspects and advantages of the present invention will becomeapparent to those skilled in the art from a study of the followingdescription of the invention and non-limiting examples.

BRIEF DESCRIPTION OF THE SEQUENCES IN THE SEQUENCE LISTING

SEQ ID NO: 1 is the native cry3A coding region.

SEQ ID NO: 2 is the amino acid sequence of the Cry3A toxin encoded bythe native cry3A gene.

SEQ ID NO: 3 is the maize optimized cry3A coding region beginning atnucleotide 144 of the native cry3A coding region.

SEQ ID NO: 4 is the amino acid sequence of the Cry3A toxin encoded bythe maize optimized cry3A gene.

SEQ ID NO: 5 is the nucleotide sequence of pCIB6850.

SEQ ID NO: 6 is the maize optimized modified cry3A054 coding sequence.

SEQ ID NO: 7 is the amino acid sequence encoded by the nucleotidesequence of SEQ ID NO: 6.

SEQ ID NO: 8 is the maize optimized modified cry3A055 coding sequence.

SEQ ID NO: 9 is the amino acid sequence encoded by the nucleotidesequence of SEQ ID NO: 8.

SEQ ID NO: 10 is the maize optimized modified cry3A085 coding sequence.

SEQ ID NO: 11 is the amino acid sequence encoded by the nucleotidesequence of SEQ ID NO: 10.

SEQ ID NO: 12 is the maize optimized modified cry3A082 coding sequence.

SEQ ID NO: 13 is the amino acid sequence encoded by the nucleotidesequence of SEQ ID NO: 12.

SEQ ID NO: 14 is the maize optimized modified cry3A058 coding sequence.

SEQ ID NO: 15 is the amino acid sequence encoded by the nucleotidesequence of SEQ ID NO: 14.

SEQ ID NO: 16 is the maize optimized modified cry3A057 coding sequence.

SEQ ID NO: 17 is the amino acid sequence encoded by the nucleotidesequence of SEQ ID NO: 16.

SEQ ID NO: 18 is the maize optimized modified cry3A056 coding sequence.

SEQ ID NO: 19 is the amino acid sequence encoded by the nucleotidesequence of SEQ ID NO: 18.

SEQ ID NO: 20 is the maize optimized modified cry3A083 coding sequence.

SEQ ID NO: 21 is the amino acid sequence encoded by the nucleotidesequence of SEQ ID NO: 20.

SEQ ID NOS: 22-34 are PCR primers useful in the present invention.

SEQ ID NO: 35 is an amino acid sequence comprising a cathepsin Grecognition site.

SEQ ID NO: 36 is an amino acid sequence comprising a cathepsin Grecognition site.

SEQ ID NO: 37 is an amino acid sequence comprising a cathepsin Grecognition site.

SEQ ID NO: 38 is an amino acid sequence comprising a cathepsin Grecognition site.

DEFINITIONS

For clarity, certain terms used in the specification are defined andpresented as follows:

“Activity” of the modified Cry3A toxins of the invention is meant thatthe modified Cry3A toxins function as orally active insect controlagents, have a toxic effect, or are able to disrupt or deter insectfeeding, which may or may not cause death of the insect. When a modifiedCry3A toxin of the invention is delivered to the insect, the result istypically death of the insect, or the insect does not feed upon thesource that makes the modified Cry3A toxin available to the insect.

“Adjacent to”—According to the present invention, an additional proteaserecognition site is “adjacent to” a naturally occurring proteaserecognition site when the additional protease recognition site is withinfour residues, preferably within three residues, more preferably withintwo residues, and most preferably within one residue of a naturallyoccurring protease recognition site. For example, an additional proteaserecognition site inserted between Pro-154 and Arg-158 of the deducedamino acid sequence of a Cry3A toxin (SEQ ID NO: 2) is “adjacent to” thenaturally occurring trypsin recognition site located between Arg-158 andAsn-159 of the deduced amino acid sequence of the Cry3A toxin (SEQ IDNO: 2).

The phrase “approximately the same position” as used herein to describethe location where an additional protease recognition site is insertedinto a Cry3A toxin in relation to a naturally occurring proteaserecognition site, means that the location is at most four residues awayfrom a naturally occurring protease recognition site. The location canalso be three or two residues away from a naturally occurring proteaserecognition site. The location can also be one residue away from anaturally occurring protease recognition site. “Approximately the sameposition” can also mean that the additional protease recognition site isinserted within a naturally occurring protease recognition site.

“Associated with/operatively linked” refer to two nucleic acid sequencesthat are related physically or functionally. For example, a promoter orregulatory DNA sequence is said to be “associated with” a DNA sequencethat codes for an RNA or a protein if the two sequences are operativelylinked, or situated such that the regulatory DNA sequence will affectthe expression level of the coding or structural DNA sequence.

A “chimeric gene” or “chimeric construct” is a recombinant nucleic acidsequence in which a promoter or regulatory nucleic acid sequence isoperatively linked to, or associated with, a nucleic acid sequence thatcodes for an mRNA or which is expressed as a protein, such that theregulatory nucleic acid sequence is able to regulate transcription orexpression of the associated nucleic acid coding sequence. Theregulatory nucleic acid sequence of the chimeric gene is not normallyoperatively linked to the associated nucleic acid sequence as found innature.

A “coding sequence” is a nucleic acid sequence that is transcribed intoRNA such as mRNA, rRNA, tRNA, snRNA, sense RNA or antisense RNA.Preferably the RNA is then translated in an organism to produce aprotein.

To “control” insects means to inhibit, through a toxic effect, theability of insect pests to survive, grow, feed, and/or reproduce, or tolimit insect-related damage or loss in crop plants. To “control” insectsmay or may not mean killing the insects, although it preferably meanskilling the insects.

Corresponding to: in the context of the present invention,“corresponding to” means that when the amino acid sequences of variantCry3A δ-endotoxins are aligned with each other, the amino acids that“correspond to” certain enumerated positions in the present inventionare those that align with these positions in the Cry3A toxin (SEQ ID NO:2), but that are not necessarily in these exact numerical positionsrelative to the particular Cry3A amino acid sequence of the invention.For example, the maize optimized cry3A gene (SEQ ID NO: 3) of theinvention encodes a Cry3A toxin (SEQ ID NO: 4) that begins at Met-48 ofthe Cry3A toxin (SEQ ID NO: 2) encoded by the native cry3A gene (SEQ IDNO: 1). Therefore, according to the present invention, amino acidnumbers 107-115, including all numbers in between, and 536-541,including all numbers in between, of SEQ ID NO: 4 correspond to aminoacid numbers 154-163, and all numbers in between, and 583-588, and allnumbers in between, respectively, of SEQ ID NO: 2.

A “Cry3A toxin”, as used herein, refers to an approximately 73 kDaBacillus thuringiensis var. tenebrionis (Kreig et al., 1983, Z. Angew.Entomol. 96:500-508) (Bt) coleopteran-active protein (Sekar et al.,1987, Proc. Natl. Acad. Sci. 84:7036-7040), for example SEQ ID NO: 2, aswell as any truncated lower molecular weight variants, derivable from aCry3A toxin, for example SEQ ID NO: 4, and retaining substantially thesame toxicity as the Cry3A toxin. The lower molecular weight variantscan be obtained by protease cleavage of naturally occurring proteaserecognition sites of the Cry3A toxin or by a second translationalinitiation codon in the same frame as the translational initiation codoncoding for the 73 kDa Cry3A toxin. The amino acid sequence of a Cry3Atoxin and the lower molecular weight variants thereof can be found in atoxin naturally occurring in Bt. A Cry3A toxin can be encoded by anative Bt gene as in SEQ ID NO: 1 or by a synthetic coding sequence asin SEQ ID NO: 3. A “Cry3A toxin” does not have any additional proteaserecognition sites over the protease recognition sites that naturallyoccur in the Cry3A toxin. A Cry3A toxin can be isolated, purified orexpressed in a heterologous system.

A “cry3A gene”, as used herein, refers to the nucleotide sequence of SEQID NO: 1 or SEQ ID NO: 3. A cry3A gene (Sekar et al., 1987, Proc. Natl.Acad. Sci. 84:7036-7040) can be naturally occurring, as found inBacillus thuringiensis var. tenebrionis (Kreig et al., 1983, Z. Angew.Entomol. 96:500-508), or synthetic and encodes a Cry3A toxin. The cry3Agene of this invention can be referred to as the native cry3A gene as inSEQ ID NO: 1 or the maize-optimized cry3A gene as in SEQ ID NO: 3.

To “deliver” a toxin means that the toxin comes in contact with aninsect, resulting in toxic effect and control of the insect. The toxincan be delivered in many recognized ways, e.g., orally by ingestion bythe insect or by contact with the insect via transgenic plantexpression, formulated protein composition(s), sprayable proteincomposition(s), a bait matrix, or any other art-recognized toxindelivery system.

“Effective insect-controlling amount” means that concentration of toxinthat inhibits, through a toxic effect, the ability of insects tosurvive, grow, feed and/or reproduce, or to limit insect-related damageor loss in crop plants. “Effective insect-controlling amount” may or maynot mean killing the insects, although it preferably means killing theinsects.

“Expression cassette” as used herein means a nucleic acid sequencecapable of directing expression of a particular nucleotide sequence inan appropriate host cell, comprising a promoter operably linked to thenucleotide sequence of interest which is operably linked to terminationsignals. It also typically comprises sequences required for propertranslation of the nucleotide sequence. The expression cassettecomprising the nucleotide sequence of interest may be chimeric, meaningthat at least one of its components is heterologous with respect to atleast one of its other components. The expression cassette may also beone that is naturally occurring but has been obtained in a recombinantform useful for heterologous expression. Typically, however, theexpression cassette is heterologous with respect to the host, i.e., theparticular nucleic acid sequence of the expression cassette does notoccur naturally in the host cell and must have been introduced into thehost cell or an ancestor of the host cell by a transformation event. Theexpression of the nucleotide sequence in the expression cassette may beunder the control of a constitutive promoter or of an inducible promoterthat initiates transcription only when the host cell is exposed to someparticular external stimulus. In the case of a multicellular organism,such as a plant, the promoter can also be specific to a particulartissue, or organ, or stage of development.

A “gene” is a defined region that is located within a genome and that,besides the aforementioned coding nucleic acid sequence, comprisesother, primarily regulatory, nucleic acid sequences responsible for thecontrol of the expression, that is to say the transcription andtranslation, of the coding portion. A gene may also comprise other 5′and 3′ untranslated sequences and termination sequences. Furtherelements that may be present are, for example, introns.

“Gene of interest” refers to any gene which, when transferred to aplant, confers upon the plant a desired characteristic such asantibiotic resistance, virus resistance, insect resistance, diseaseresistance, or resistance to other pests, herbicide tolerance, improvednutritional value, improved performance in an industrial process oraltered reproductive capability. The “gene of interest” may also be onethat is transferred to plants for the production of commerciallyvaluable enzymes or metabolites in the plant.

A “gut protease” is a protease naturally found in the digestive tract ofan insect. This protease is usually involved in the digestion ofingested proteins.

A “heterologous” nucleic acid sequence is a nucleic acid sequence notnaturally associated with a host cell into which it is introduced,including non-naturally occurring multiple copies of a naturallyoccurring nucleic acid sequence.

A “homologous” nucleic acid sequence is a nucleic acid sequencenaturally associated with a host cell into which it is introduced.

“Homologous recombination” is the reciprocal exchange of nucleic acidfragments between homologous nucleic acid molecules.

“Insecticidal” is defined as a toxic biological activity capable ofcontrolling insects, preferably by killing them.

A nucleic acid sequence is “isocoding with” a reference nucleic acidsequence when the nucleic acid sequence encodes a polypeptide having thesame amino acid sequence as the polypeptide encoded by the referencenucleic acid sequence.

An “isolated” nucleic acid molecule or an isolated toxin is a nucleicacid molecule or toxin that, by the hand of man, exists apart from itsnative environment and is therefore not a product of nature. An isolatednucleic acid molecule or toxin may exist in a purified form or may existin a non-native environment such as, for example, a recombinant hostcell.

A “modified Cry3A toxin” of this invention, refers to a Cry3A-derivedtoxin having at least one additional protease recognition site that isrecognized by a gut protease of a target insect, which does notnaturally occur in a Cry3A toxin. A modified Cry3A toxin is notnaturally occurring and, by the hand of man, comprises an amino acidsequence that is not identical to a naturally occurring toxin found inBacillus thuringiensis. The modified Cry3A toxin causes higher mortalityto a target insect than the mortality caused by a Cry3A toxin to thesame target insect.

A “modified cry3A gene” according to this invention, refers to acry3A-derived gene comprising the coding sequence of at least oneadditional protease recognition site that does not naturally occur in anunmodified cry3A gene. The modified cry3A gene can be derived from anative cry3A gene or from a synthetic cry3A gene.

A “naturally occurring protease recognition site” is a location within aCry3A toxin that is cleaved by a non-insect derived protease or by aprotease or gut extract from an insect species susceptible to the Cry3Atoxin. For example, a naturally occurring protease recognition site,recognized by trypsin and proteases found in a susceptible insect gutextract, exists between Arg-158 and Asn-159 of the deduced Cry3A toxinamino acid sequence (SEQ ID NO: 2). Naturally occurring proteaserecognition sites, recognized by chymotrypsin, exist between His-161 andSer-162 as well as between Tyr-587 and Tyr-588 of the deduced Cry3Atoxin amino acid sequence (SEQ ID NO: 2).

A “nucleic acid molecule” or “nucleic acid sequence” is a linear segmentof single- or double-stranded DNA or RNA that can be isolated from anysource. In the context of the present invention, the nucleic acidmolecule is preferably a segment of DNA.

A “plant” is any plant at any stage of development, particularly a seedplant.

A “plant cell” is a structural and physiological unit of a plant,comprising a protoplast and a cell wall. The plant cell may be in theform of an isolated single cell or a cultured cell, or as a part of ahigher organized unit such as, for example, plant tissue, a plant organ,or a whole plant.

“Plant cell culture” means cultures of plant units such as, for example,protoplasts, cell culture cells, cells in plant tissues, pollen, pollentubes, ovules, embryo sacs, zygotes and embryos at various stages ofdevelopment.

“Plant material” refers to leaves, stems, roots, flowers or flowerparts, fruits, pollen, egg cells, zygotes, seeds, cuttings, cell ortissue cultures, or any other part or product of a plant.

A “plant organ” is a distinct and visibly structured and differentiatedpart of a plant such as a root; stem, leaf, flower bud, or embryo.

“Plant tissue” as used herein means a group of plant cells organizedinto a structural and functional unit. Any tissue of a plant in plantaor in culture is included. This term includes, but is not limited to,whole plants, plant organs, plant seeds, tissue culture and any groupsof plant cells organized into structural and/or functional units. Theuse of this term in conjunction with, or in the absence of, any specifictype of plant tissue as listed above or otherwise embraced by thisdefinition is not intended to be exclusive of any other type of planttissue.

A “promoter” is an untranslated DNA sequence upstream of the codingregion that contains the binding site for RNA polymerase and initiatestranscription of the DNA. The promoter region may also include otherelements that act as regulators of gene expression.

A “protoplast” is an isolated plant cell without a cell wall or withonly parts of the cell wall.

“Regulatory elements” refer to sequences involved in controlling theexpression of a nucleotide sequence. Regulatory elements comprise apromoter operably linked to the nucleotide sequence of interest andtermination signals. They also typically encompass sequences requiredfor proper translation of the nucleotide sequence.

“Replaces” a naturally occurring protease recognition site—According tothe present invention, an additional protease recognition site“replaces” a naturally occurring protease recognition site wheninsertion of the additional protease recognition site eliminates thenaturally occurring protease recognition site. For example, anadditional protease recognition site inserted between Pro-154 andPro-160 of the deduced amino acid sequence of a Cry3A toxin (SEQ ID NO:2) which eliminates the Arg-158 and Asn-159 residues “replaces” thenaturally occurring trypsin recognition site located between Arg-158 andAsn-159 of the deduced amino acid sequence of the Cry3A toxin (SEQ ID.NO: 2).

“Serine proteases”, describe the same group of enzymes that catalyze thehydrolysis of covalent peptidic bonds using a mechanism based onnucleophilic attack of the targeted peptidic bond by a serine. Serineproteases are sequence specific. That is each serine protease recognizesa specific sub-sequence within a protein where enzymatic recognitionoccurs.

A “target insect” is an insect pest species that has little or nosusceptibility to a Cry3A toxin and is identified as being a candidatefor using the technology of the present invention to control. Thiscontrol can be achieved through several means but most preferablythrough the expression of the nucleic acid molecules of the invention intransgenic plants.

A “target insect gut protease” is a protease found in the gut of atarget insect whose recognition site can be inserted into a Cry3A toxinto create a modified Cry3A toxin of the invention.

“Transformation” is a process for introducing heterologous nucleic acidinto a host cell or organism. In particular, “transformation” means thestable integration of a DNA molecule into the genome of an organism ofinterest.

“Transformed/transgenic/recombinant” refer to a host organism such as abacterium or a plant into which a heterologous nucleic acid molecule hasbeen introduced. The nucleic acid molecule can be stably integrated intothe genome of the host or the nucleic acid molecule can also be presentas an extrachromosomal molecule. Such an extrachromosomal molecule canbe auto-replicating. Transformed cells, tissues, or plants areunderstood to encompass not only the end product of a transformationprocess, but also transgenic progeny thereof. A “non-transformed”,“non-transgenic”, or “non-recombinant” host refers to a wild-typeorganism, e.g., a bacterium or plant, which does not contain theheterologous nucleic acid molecule.

“Within” a naturally occurring protease recognition site—According tothe present invention, an additional protease recognition site is“within” a naturally occurring protease recognition site when theadditional protease recognition site lies between the amino acid residuethat comes before and the amino acid residue that comes after thenaturally occurring protease recognition site. For example, anadditional protease recognition site inserted between Tyr-587 andTyr-588 of the deduced amino acid sequence of a Cry3A toxin (SEQ ID NO:2) is “within” a naturally occurring chymotrypsin recognition sitelocated between Tyr-587 and Tyr-588 of the deduced amino acid sequenceof the Cry3A toxin (SEQ ID NO: 2). The insertion of an additionalprotease recognition site within a naturally occurring proteaserecognition site may or may not change the recognition of the naturallyoccurring protease recognition site by a protease.

Nucleotides are indicated by their bases by the following standardabbreviations: adenine (A), cytosine (C), thymine (T), and guanine (O).Amino acids are likewise indicated by the following standardabbreviations: alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N),aspartic acid (Asp; D), cysteine (Cys; C), glutamine (Gln; Q), glutamicacid (Glu; E), glycine (Gly; G), histidine (His; H), isoleucine (Ile;1), leucine (Lou; L), lysine (Lys; K), methionine (Met; M),phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine(Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).

DESCRIPTION

This invention relates to modified cry3A nucleic acid sequences whoseexpression results in modified Cry3A toxins, and to the making and usingof the modified Cry3A toxins to control insect pests. The expression ofthe modified cry3A nucleic acid sequences results in modified Cry3Atoxins that can be used to control coleopteran insects such as westerncorn rootworm and northern corn rootworm. A modified Cry3A toxin of thepresent invention comprises at least one additional protease recognitionsite that does not naturally occur in a Cry3A toxin. The additionalprotease recognition site, which is recognized by a gut protease of atarget insect, is inserted at approximately the same position as anaturally occurring protease recognition site in a Cry3A toxin. Themodified Cry3A toxin causes higher mortality to a target insect than themortality caused by a Cry3A toxin to the same target insect. Preferably,the modified Cry3A toxin causes at least about 50% mortality to thetarget insect to which a Cry3A toxin causes up to about 30% mortality.

In one preferred embodiment, the invention encompasses an isolatednucleic acid molecule that encodes a modified Cry3A toxin, wherein theadditional protease recognition site is recognized by the target insectgut protease, cathepsin G. Cathepsin G activity is determined to bepresent in the gut of the target insect, western corn rootworm, asdescribed in Example 2. Preferably, the substrate amino acid sequence,AAPF (SEQ ID NO: 35), used to determine the presence of the cathepsin Gactivity is inserted into the Cry3A toxin according to the presentinvention. Other cathepsin G recognition sites can also be usedaccording to the present invention, for example, AAPM (SEQ ID NO: 36),AVPF (SEQ ID NO: 37), PFLF (SEQ ID NO: 38) or other cathepsin Grecognition sites as determined by the method of Tanaka et al., 1985(Biochemistry 24:2040-2047), incorporated herein by reference. Proteaserecognition sites of other proteases identified in a target insect gutcan be used, for example, protease recognition sites recognized by otherserine proteases, cysteine proteases and aspartic proteases. Preferableserine proteases encompassed by this embodiment include trypsin,chymotrypsin, carboxypeptidase, endopeptidase and elastase.

In another preferred embodiment, the invention encompasses an isolatednucleic acid molecule that encodes a modified Cry3A toxin wherein theadditional protease recognition site is inserted in either domain I ordomain III or in both domain I and domain III of the Cry3A toxin.Preferably, the additional protease recognition site is inserted indomain I, domain III, or domain I and domain III at a position thatreplaces, is adjacent to, or is within a naturally occurring proteaserecognition site in the Cry3A toxin. Specifically exemplified herein arenucleic acid molecules that encode modified Cry3A toxins that comprise acathepsin G recognition site inserted in domain I, domain III, or domainI and domain III at a position that replaces, is adjacent to, or iswithin a naturally occurring protease recognition site in the unmodifiedCry3A toxin.

Specifically exemplified teachings of methods to make modified cry3Anucleic acid molecules that encode modified Cry3A toxins can be found inExample 3. Those skilled in the art will recognize that other methodsknown in the art can also be used to insert additional proteaserecognition sites into Cry3A toxins according to the present invention.

In another preferred embodiment, the invention encompasses an isolatednucleic acid molecule that encodes a modified Cry3A toxin wherein theadditional protease recognition site is inserted in domain I betweenamino acids corresponding to amino acid numbers 154 and 162 of SEQ IDNO: 2. Preferably, the additional protease recognition site is insertedbetween amino acid numbers 154 and 162 of SEQ ID NO: 2 or between aminoacid numbers 107 and 115 of SEQ ID NO: 4. In a preferred embodiment, theadditional protease recognition site is inserted between amino acidscorresponding to amino acid numbers 154 and 160 of SEQ ID NO: 2.Preferably, the additional protease recognition site is inserted betweenamino acid number 154 and 160 of SEQ ID NO: 2 or between amino acidnumbers 107 and 113 of SEQ ID NO: 4. Specifically exemplified herein isa nucleic acid molecule, designated cry3A054 (SEQ ID NO: 6), thatencodes the modified Cry3A054 toxin (SEQ ID NO: 7) comprising acathepsin G recognition site inserted in domain I between amino acidnumbers 107 and 113 of SEQ ID NO: 4. The cathepsin G recognition sitereplaces a naturally occurring trypsin recognition site and is adjacentto a naturally occurring chymotrypsin recognition site. When expressedin a heterologous host, the nucleic acid molecule of SEQ ID NO: 6results in insect control activity against western corn rootworm andnorthern corn rootworm, showing that the nucleic acid sequence set forthin SEQ ID NO: 6 is sufficient for such insect control activity.

In another preferred embodiment, the additional protease recognitionsite is inserted in domain I between amino acids corresponding to aminoacid numbers 154 and 158 of SEQ ID NO: 2. Preferably, the additionalprotease recognition site is inserted in domain I between amino acidnumbers 154 and 158 of SEQ ID NO: 2 or between amino acid numbers 107and 111 of SEQ ID NO: 4. Specifically exemplified herein are nucleicacid molecules, designated cry3A055 (SEQ ID NO: 8), that encodes themodified Cry3A055 toxin (SEQ ID NO: 9), and cry3A085 (SEQ ID NO: 10),that encodes the modified Cry3A085 toxin (SEQ ID NO: 11), comprising acathepsin G recognition site inserted in domain I between amino acidnumbers 107 and 111 of SEQ ID NO: 4. The cathepsin G recognition site isadjacent to naturally occurring trypsin and chymotrypsin recognitionsites. When expressed in a heterologous host, the nucleic acid moleculeof SEQ ID NO: 8 or SEQ ID NO: 10 results in insect control activityagainst western corn rootworm and northern corn rootworm, showing thatthe nucleic acid sequence set forth in SEQ ID NO: 8 or SEQ ID NO: 10 issufficient for such insect control activity.

In a preferred embodiment, the invention encompasses an isolated nucleicacid molecule that encodes a modified Cry3A toxin wherein the additionalprotease recognition site is inserted in domain III between amino acidscorresponding to amino acid numbers 583 and 589 of SEQ ID NO: 2.Preferably, the additional protease site is inserted in domain IIIbetween amino acid numbers 583 and 589 of SEQ ID NO: 2 or between aminoacid numbers 536 and 542 of SEQ ID NO: 4.

In another preferred embodiment, the invention encompasses an isolatednucleic acid molecule that encodes a modified Cry3A toxin wherein theadditional protease recognition site is inserted in domain III betweenamino acids corresponding to amino acid numbers 583 and 588 of SEQ IDNO: 2. Preferably, the additional protease site is inserted in domainIII between amino acid numbers 583 and 588 of SEQ ID NO: 2 or betweenamino acid numbers 536 and 541 of SEQ ID NO: 4. Specifically exemplifiedherein is a nucleic acid molecule, designated cry3A082 (SEQ ID NO: 12),that encodes the modified Cry3A082 toxin (SEQ ID NO: 13) comprising acathepsin G recognition site inserted in domain III between amino acidnumbers 536 and 541 of SEQ ID NO: 4. The cathepsin G recognition sitereplaces a naturally occurring chymotrypsin recognition site. Whenexpressed in a heterologous host, the nucleic acid molecule of SEQ IDNO: 12 results in insect control activity against western corn rootwormand northern corn rootworm, showing that the nucleic acid sequence setforth in SEQ ID NO: 12 is sufficient for such insect control activity.

In another preferred embodiment, the additional protease recognitionsite is inserted in domain III between amino acids corresponding toamino acid numbers 587 and 588 of SEQ ID NO: 2. Preferably, theadditional protease site is inserted in domain III between amino acidnumbers 587 and 588 of SEQ ID NO: 2 or between amino acid numbers 540and 541 of SEQ ID NO: 4. Specifically exemplified herein is a nucleicacid molecule, designated cry3A058 (SEQ ID NO: 14), that encodes themodified Cry3A058 toxin (SEQ ID NO: 15) comprising a cathepsin Grecognition site inserted in domain III between amino acid numbers 540and 541 of SEQ ID NO: 4. The cathepsin G recognition site is within anaturally occurring chymotrypsin recognition site. When expressed in aheterologous host, the nucleic acid molecule of SEQ ID NO: 14 results ininsect control activity against western corn rootworm and northern cornrootworm, showing that the nucleic acid sequence set forth in SEQ ID NO:14 is sufficient for such insect control activity.

In yet another preferred embodiment, the invention encompasses anisolated nucleic acid molecule that encodes a modified Cry3A toxinwherein the additional protease recognition site is inserted in domain Ibetween amino acids corresponding to amino acid numbers 154 and 160 andin domain III between amino acids corresponding to amino acid numbers587 and 588 of SEQ ID NO: 2. Preferably, the additional proteaserecognition site is inserted in domain I between amino acid numbers 154and 160 and in domain III between amino acid numbers 587 and 588 of SEQID NO: 2 or in domain I between amino acid numbers 107 and 113 and indomain III between amino acid numbers 540 and 541 of SEQ ID NO: 4.Specifically exemplified herein is a nucleic acid molecule, designatedcry3A057 (SEQ ID NO: 16), that encodes the modified Cry3A057 toxin (SEQID NO: 17) comprising a cathepsin G recognition site inserted in domainI between amino acid numbers 107 and 113 and in domain III between aminoacid numbers 540 and 541 of SEQ ID NO: 4. The cathepsin G recognitionsite replaces a naturally occurring trypsin recognition site and isadjacent to a naturally occurring chymotrypsin recognition site indomain I and is within a naturally occurring chymotrypsin recognitionsite in domain III. When expressed in a heterologous host, the nucleicacid molecule of SEQ ID NO: 16 results in insect control activityagainst western corn rootworm and northern corn rootworm, showing thatthe nucleic acid sequence set forth in SEQ ID NO: 16 is sufficient forsuch insect control activity.

In yet another preferred embodiment, the additional protease recognitionsite is located in domain I between amino acids corresponding to aminoacid numbers 154 and 158 and in domain III between amino acidscorresponding to amino acid numbers 587 and 588 of SEQ ID NO: 2.Preferably, the additional protease recognition site is inserted indomain I between amino acid numbers 154 and 158 and in domain IIIbetween amino acid numbers 587 and 588 of SEQ ID NO: 2 or in domain Ibetween amino acid numbers 107 and 111 and in domain III between aminoacid numbers 540 and 541 of SEQ ID NO: 4. Specifically exemplifiedherein is the nucleic acid molecule designated cry3A056 (SEQ ID NO: 18),which encodes the modified Cry3A056 toxin (SEQ ID NO: 19) comprising acathepsin G recognition site inserted in domain I between amino acidnumbers 107 and 111 and in domain III between amino acid numbers 540 and541 of SEQ ID NO: 4. The cathepsin G recognition site is adjacent tonaturally occurring trypsin and chymotrypsin recognition sites in domainI and is within a naturally occurring chymotrypsin recognition site indomain III. When expressed in a heterologous host, the nucleic acidmolecule of SEQ ID NO: 18 results in insect control activity againstwestern corn rootworm and northern corn rootworm, showing that thenucleic acid sequence set forth in SEQ ID NO: 18 is sufficient for suchinsect control activity.

In still another preferred embodiment, the additional proteaserecognition site is located in domain I between amino acidscorresponding to amino acid numbers 154 and 158 and in domain IIIbetween amino acids corresponding to amino acid numbers 583 and 588 ofSEQ ID NO: 2. Preferably, the additional protease recognition site isinserted in domain I between amino acid numbers 154 and 158 and indomain III between amino acid numbers 583 and 588 of SEQ ID NO: 2 or indomain I between amino acid numbers 107 and 111 and in domain IIIbetween amino acid numbers 536 and 541 of SEQ ID NO: 4. Specificallyexemplified herein is a nucleic acid molecule, designated cry3A083 (SEQID NO: 20), which encodes the modified Cry3A083 toxin (SEQ ID NO: 21)comprising a cathepsin G recognition site inserted in domain I betweenamino acid numbers 107 and 111 and in domain III between amino acidnumbers 536 and 541 of SEQ ID NO: 4. The cathepsin G recognition site isadjacent to naturally occurring trypsin and chymotrypsin recognitionsites in domain I and replaces a naturally occurring chymotrypsinrecognition site in domain III. When expressed in a heterologous host,the nucleic acid molecule of SEQ ID NO: 20 results in insect controlactivity against western corn rootworm and northern corn rootworm,showing that the nucleic acid sequence set forth in SEQ ID NO: 20 issufficient for such insect control activity.

In a preferred embodiment, the isolated nucleic acid molecule of thepresent invention comprises nucleotides 1-1791 of SEQ ID NO: 6,nucleotides 1-1806 of SEQ ID NO: 8, nucleotides 1-1812 of SEQ ID NO: 10,nucleotides 1-1794 of SEQ ID NO: 12, nucleotides 1-1818 of SEQ ID NO:14, nucleotides 1-1812 of SEQ ID NO: 16, nucleotides 1-1791 of SEQ IDNO: 18, and nucleotides 1-1818 of SEQ ID NO: 20.

In another preferred embodiment, the invention encompasses the isolatednucleic acid molecule that encodes a modified Cry3A toxin comprising theamino acid sequence set forth in SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO:11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, or SEQID NO: 21.

The present invention also encompasses recombinant vectors comprisingthe nucleic acid sequences of this invention. In such vectors, thenucleic acid sequences are preferably comprised in expression cassettescomprising regulatory elements for expression of the nucleotidesequences in a host cell capable of expressing the nucleotidessequences. Such regulatory elements usually comprise promoter andtermination signals and preferably also comprise elements allowingefficient translation of polypeptides encoded by the nucleic acidsequences of the present invention. Vectors comprising the nucleic acidsequences are usually capable of replication in particular host cells,preferably as extrachromosomal molecules, and are therefore used toamplify the nucleic acid sequences of this invention in the host cells.In one embodiment, host cells for such vectors are microorganisms, suchas bacteria, in particular Bacillus thuringiensis or E. coli. In anotherembodiment, host cells for such recombinant vectors are endophytes orepiphytes. A preferred host cell for such vectors is a eukaryotic cell,such as a plant cell. Plant cells such as maize cells are most preferredhost cells. In another preferred embodiment, such vectors are viralvectors and are used for replication of the nucleotide sequences inparticular host cells, e.g. insect cells or plant cells. Recombinantvectors are also used for transformation of the nucleotide sequences ofthis invention into host cells, whereby the nucleotide sequences arestably integrated into the DNA of such host cells. In one, such hostcells are prokaryotic cells. In a preferred embodiment, such host cellsare eukaryotic cells, such as plant cells. In a most preferredembodiment, the host cells are plant cells, such as maize cells.

In another aspect, the present invention encompasses modified Cry3Atoxins produced by the expression of the nucleic acid molecules of thepresent invention.

In preferred embodiments, the modified Cry3A toxins of the inventioncomprise a polypeptide encoded by a nucleotide sequence of theinvention. In a further preferred embodiment, the modified Cry3A toxinis produced by the expression of the nucleic acid molecule comprisingnucleotides 1-1791 of SEQ ID NO: 6, nucleotides 1-1806 of SEQ ID NO: 8,nucleotides 1-1812 of SEQ ID NO: 10, nucleotides 1-1794 of SEQ ID NO:12, nucleotides 1-1818 of SEQ ID NO: 14, nucleotides 1-1812 of SEQ IDNO: 16, nucleotides 1-1791 of SEQ ID NO: 18, and nucleotides 1-1818 ofSEQ ID NO: 20.

In a preferred embodiment, a modified Cry3A toxin of the presentinvention comprises the amino acid sequence set forth in SEQ ID NO: 7,SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO:17, SEQ ID NO: 19, or SEQ ID NO: 21.

The modified Cry3A toxins of the present invention have insect controlactivity when tested against insect pests in bioassays. In anotherpreferred embodiment, the modified Cry3A toxins of the invention areactive against coleopteran insects, preferably against western cornrootworm and northern corn rootworm. The insect controlling propertiesof the modified Cry3A toxins of the invention are further illustrated inExamples 4 and 6.

The present invention also encompasses a composition comprising aneffective insect-controlling amount of a modified Cry3A toxin accordingto the invention.

In another preferred embodiment, the invention encompasses a method ofproducing a modified Cry3A toxin that is active against insects,comprising: (a) obtaining a host cell comprising a chimeric gene, whichitself comprises a heterologous promoter sequence operatively linked tothe nucleic acid molecule of the invention; and (b) expressing thenucleic acid molecule in the transgenic host cell, which results in atleast one modified Cry3A toxin that is active against insects.

In a further preferred embodiment, the invention encompasses a method ofproducing an insect-resistant transgenic plant, comprising introducing anucleic acid molecule of the invention into the transgenic plant,wherein the nucleic acid molecule is expressible in the transgenic plantin an effective amount to control insects. In a preferred embodiment,the insects are coleopteran insects, preferably western corn rootwormand northern corn rootworm.

In yet a further preferred embodiment, the invention encompasses amethod of controlling insects, comprising delivering to the insects aneffective amount of a modified Cry3A toxin of the invention. Accordingto this embodiment, the insects are coleopteran insects, preferably,western corn rootworm and northern corn rootworm. Preferably, themodified Cry3A toxin is delivered to the insects orally. In onepreferred aspect, the toxin is delivered orally through a transgenicplant comprising a nucleic acid sequence that expresses a modified Cry3Atoxin of the present invention.

The present invention also encompasses a method of making a modifiedCry3A toxin, comprising: (a) obtaining a cry3A toxin gene which encodesa Cry3A toxin; (b) identifying a gut protease of a target insect; (c)obtaining a nucleotide sequence which encodes a recognition site for thegut protease; (d) inserting the nucleotide sequence of (c) into eitherdomain I or domain III or both domain I and domain III at a positionthat replaces, is within, or adjacent to a nucleotide sequence thatcodes for a naturally occurring protease recognition site in the cry3Atoxin gene, thus creating a modified cry3A toxin gene; (e) inserting themodified cry3A toxin gene in an expression cassette; (f) expressing themodified cry3A toxin gene in a non-human host cell, resulting in thehost cell producing a modified Cry3A toxin; and, (g) bioassaying themodified Cry3A toxin against a target insect, which causes highermortality to the target insect than the mortality caused by a Cry3Atoxin. In a preferred embodiment, the modified Cry3A toxin causes atleast about 50% mortality to the target insect when the Cry3A toxincauses up to about 30% mortality.

The present invention further encompasses a method of controllinginsects wherein the transgenic plant further comprises a second nucleicacid sequence or groups of nucleic acid sequences that encode a secondpesticidal principle. Particularly preferred second nucleic acidsequences are those that encode a δ-endotoxin, those that encode aVegetative Insecticidal Protein toxin, disclosed in U.S. Pat. Nos.5,849,870 and 5,877,012, incorporated herein by reference, or those thatencode a pathway for the production of a non-proteinaceous principle.

In further embodiments, the nucleotide sequences of the invention can befurther modified by incorporation of random mutations in a techniqueknown as in vitro recombination or DNA shuffling. This technique isdescribed in Stemmer et al., Nature 370:389-391 (1994) and U.S. Pat. No.5,605,793, which are incorporated herein by reference. Millions ofmutant copies of a nucleotide sequence are produced based on an originalnucleotide sequence of this invention and variants with improvedproperties, such as increased insecticidal activity, enhanced stability,or different specificity or ranges of target-insect pests are recovered.The method encompasses forming a mutagenized double-strandedpolynucleotide from a template double-stranded polynucleotide comprisinga nucleotide sequence of this invention, wherein the templatedouble-stranded polynucleotide has been cleaved intodouble-stranded-random fragments of a desired size, and comprises thesteps of adding to the resultant population of double-stranded randomfragments one or more single or double-stranded oligonucleotides,wherein said oligonucleotides comprise an area of identity and an areaof heterology to the double-stranded template polynucleotide; denaturingthe resultant mixture of double-stranded random fragments andoligonucleotides into single-stranded fragments; incubating theresultant population of single-stranded fragments with a polymeraseunder conditions which result in the annealing of said single-strandedfragments at said areas of identity to form pairs of annealed fragments,said areas of identity being sufficient for one member of a pair toprime replication of the other, thereby forming a mutagenizeddouble-stranded polynucleotide; and repeating the second and third stepsfor at least two further cycles, wherein the resultant mixture in thesecond step of a further cycle includes the mutagenized double-strandedpolynucleotide from the third step of the previous cycle, and thefurther cycle forms a further mutagenized double-strandedpolynucleotide. In a preferred embodiment, the concentration of a singlespecies of double-stranded random fragment in the population ofdouble-stranded random fragments is less than 1% by weight of the totalDNA. In a further preferred embodiment, the template double-strandedpolynucleotide comprises at least about 100 species of polynucleotides.In another preferred embodiment, the size of the double-stranded randomfragments is from about 5 bp to 5 kb. In a further preferred embodiment,the fourth step of the method comprises repeating the second and thethird steps for at least 10 cycles.

Expression of the Nucleotide Sequences in Heterologous Microbial Hosts

As biological insect control agents, the insecticidal modified Cry3Atoxins are produced by expression of the nucleotide sequences inheterologous host cells capable of expressing the nucleotide sequences.In a first embodiment, B. thuringiensis cells comprising modificationsof a nucleotide sequence of this invention are made. Such modificationsencompass mutations or deletions of existing regulatory elements, thusleading to altered expression of the nucleotide sequence, or theincorporation of new regulatory elements controlling the expression ofthe nucleotide sequence. In another embodiment, additional copies of oneor more of the nucleotide sequences are added to Bacillus thuringiensiscells either by insertion into the chromosome or by introduction ofextrachromosomally replicating molecules containing the nucleotidesequences.

In another embodiment, at least one of the nucleotide sequences of theinvention is inserted into an appropriate expression cassette,comprising a promoter and termination signal. Expression of thenucleotide sequence is constitutive, or an inducible promoter respondingto various types of stimuli to initiate transcription is used. In apreferred embodiment, the cell in which the toxin is expressed is amicroorganism, such as a virus, bacteria, or a fungus. In a preferredembodiment, a virus, such as a baculovirus, contains a nucleotidesequence of the invention in its genome and expresses large amounts ofthe corresponding insecticidal toxin after infection of appropriateeukaryotic cells that are suitable for virus replication and expressionof the nucleotide sequence. The insecticidal toxin thus produced is usedas an insecticidal agent. Alternatively, baculoviruses engineered toinclude the nucleotide sequence are used to infect insects in vivo andkill them either by expression of the insecticidal toxin or by acombination of viral infection and expression of the insecticidal toxin.

Bacterial cells are also hosts for the expression of the nucleotidesequences of the invention. In a preferred embodiment, non-pathogenicsymbiotic bacteria, which are able to live and replicate within planttissues, so-called endophytes, or non-pathogenic symbiotic bacteria,which are capable of colonizing the phyllosphere or the rhizosphere,so-called epiphytes, are used. Such bacteria include bacteria of thegenera Agrobacterium, Alcaligenes, Azospirillum, Azotobacter, Bacillus,Clavibacter, Enterobacter, Erwinia, Flavobacter, Klebsiella,Pseudomonas, Rhizobium, Serratia, Streptomyces and Xanthomonas.Symbiotic fungi, such as Trichoderma and Gliocladium are also possiblehosts for expression of the inventive nucleotide sequences for the samepurpose.

Techniques for these genetic manipulations are specific for thedifferent available hosts and are known in the art. For example, theexpression vectors pKK223-3 and pKK223-2 can be used to expressheterologous genes in E. coli, either in transcriptional ortranslational fusion, behind the tac or trc promoter. For the expressionof operons encoding multiple ORFs, the simplest procedure is to insertthe operon into a vector such as pKK223-3 in transcriptional fusion,allowing the cognate ribosome binding site of the heterologous genes tobe used. Techniques for overexpression in gram-positive species such asBacillus are also known in the art and can be used in the context ofthis invention (Quax et al. In: Industrial Microorganisms: Basic andApplied Molecular Genetics, Eds. Baltz et al., American Society forMicrobiology, Washington (1993)). Alternate systems for overexpressionrely for example, on yeast vectors and include the use of Pichia,Saccharomyces and Kluyveromyces (Sreekrishna, In: Industrialmicroorganisms: basic and applied molecular genetics, Baltz, Hegeman,and Skatrud eds., American Society for Microbiology, Washington (1993);Dequin & Barre, Biotechnology L2:173-177 (1994); van den Berg et al.,Biotechnology 8:135-139 (1990)).

Plant Transformation

In a particularly preferred embodiment, at least one of the insecticidalmodified Cry3A toxins of the invention is expressed in a higherorganism, e.g., a plant. In this case, transgenic plants expressingeffective amounts of the modified Cry3A toxins protect themselves frominsect pests. When the insect starts feeding on such a transgenic plant,it also ingests the expressed modified Cry3A toxins. This will deter theinsect from further biting into the plant tissue or may even harm orkill the insect. A nucleotide sequence of the present invention isinserted into an expression cassette, which is then preferably stablyintegrated in the genome of said plant. In another preferred embodiment,the nucleotide sequence is included in a non-pathogenic self-replicatingvirus. Plants transformed in accordance with the present invention maybe monocots or dicots and include, but are not limited to, maize, wheat,barley, rye, sweet potato, bean, pea, chicory, lettuce, cabbage,cauliflower, broccoli, turnip, radish, spinach, asparagus, onion,garlic, pepper, celery, squash, pumpkin, hemp, zucchini, apple, pear,quince, melon, plum, cherry, peach, nectarine, apricot, strawberry,grape, raspberry, blackberry, pineapple, avocado, papaya, mango, banana,soybean, tomato, sorghum, sugarcane, sugar beet, sunflower, rapeseed,clover, tobacco, carrot, cotton, alfalfa, rice, potato, eggplant,cucumber, Arabidopsis, and woody plants such as coniferous and deciduoustrees.

Once a desired nucleotide sequence has been transformed into aparticular plant species, it may be propagated in that species or movedinto other varieties of the same species, particularly includingcommercial varieties, using traditional breeding techniques.

A nucleotide sequence of this invention is preferably expressed intransgenic plants, thus causing the biosynthesis of the correspondingmodified Cry3A toxin in the transgenic plants. In this way, transgenicplants with enhanced resistance to insects are generated. For theirexpression in transgenic plants, the nucleotide sequences of theinvention may require other modifications and optimization. Although inmany cases genes from microbial organisms can be expressed in plants athigh levels without modification, low expression in transgenic plantsmay result from microbial nucleotide sequences having codons that arenot preferred in plants. It is known in the art that all organisms havespecific preferences for codon usage, and the codons of the nucleotidesequences described in this invention can be changed to conform withplant preferences, while maintaining the amino acids encoded thereby.Furthermore, high expression in plants is best achieved from codingsequences that have at least about 35% GC content, preferably more thanabout 45%, more preferably more than about 50%, and most preferably morethan about 60%. Microbial nucleotide sequences that have low GC contentsmay express poorly in plants due to the existence of ATTTA motifs thatmay destabilize messages, and AATAAA motifs that may cause inappropriatepolyadenylation. Although preferred gene sequences may be adequatelyexpressed in both monocotyledonous and dicotyledonous plant species,sequences can be modified to account for the specific codon preferencesand GC content preferences of monocotyledons or dicotyledons as thesepreferences have been shown to differ (Murray et al. Nucl. Acids Res.17:477-498 (1989)). In addition, the nucleotide sequences are screenedfor the existence of illegitimate splice sites that may cause messagetruncation. All changes required to be made within the nucleotidesequences such as those described above are made using well knowntechniques of site directed mutagenesis, PCR, and synthetic geneconstruction using the methods described in the published patentapplications EP 0 385 962 (to Monsanto), EP 0 359 472 (to Lubrizol, andWO 93/07278 (to Ciba-Geigy).

In one embodiment of the invention a cry3A gene is made according to theprocedure disclosed in U.S. Pat. No. 5,625,136, herein incorporated byreference. In this procedure, maize preferred codons, i.e., the singlecodon that most frequently encodes that amino acid in maize, are used.The maize preferred codon for a particular amino acid might be derived,for example, from known gene sequences from maize. Maize codon usage for28 genes from maize plants is found in Murray et al., Nucleic AcidsResearch 17:477-498 (1989), the disclosure of which is incorporatedherein by reference. A synthetic sequence made with maize optimizedcodons is set forth in SEQ ID NO: 3.

In this manner, the nucleotide sequences can be optimized for expressionin any plant. It is recognized that all or any part of the gene sequencemay be optimized or synthetic. That is, synthetic or partially optimizedsequences may also be used.

For efficient initiation of translation, sequences adjacent to theinitiating methionine may require modification. For example, they can bemodified by the inclusion of sequences known to be effective in plants.Joshi has suggested an appropriate consensus for plants (NAR15:6643-6653 (1987)) and Clonetech suggests a further consensustranslation initiator (1993/1994 catalog, page 210). These consensusesare suitable for use with the nucleotide sequences of this invention.The sequences are incorporated into constructions comprising thenucleotide sequences, up to and including the ATG (whilst leaving thesecond amino acid unmodified), or alternatively up to and including theGTC subsequent to the ATG (with the possibility of modifying the secondamino acid of the transgene).

Expression of the nucleotide sequences in transgenic plants is driven bypromoters that function in plants. The choice of promoter will varydepending on the temporal and spatial requirements for expression, andalso depending on the target species. Thus, expression of the nucleotidesequences of this invention in leaves, in stalks or stems, in ears, ininflorescences (e.g. spikes, panicles, cobs, etc.), in roots, and/orseedlings is preferred. In many cases, however, protection against morethan one type of insect pest is sought, and thus expression in multipletissues is desirable. Although many promoters from dicotyledons havebeen shown to be operational in monocotyledons and vice versa, ideallydicotyledonous promoters are selected for expression in dicotyledons,and monocotyledonous promoters for expression in monocotyledons.However, there is no restriction to the provenance of selectedpromoters; it is sufficient that they are operational in driving theexpression of the nucleotide sequences in the desired cell.

Preferred promoters that are expressed constitutively include promotersfrom genes encoding actin or ubiquitin and the CaMV 35S and 19Spromoters. The nucleotide sequences of this invention can also beexpressed under the regulation of promoters that are chemicallyregulated. This enables the insecticidal modified Cry3A toxins to besynthesized only when the crop plants are treated with the inducingchemicals. Preferred technology for chemical induction of geneexpression is detailed in the published application EP 0 332 104 (toCiba-Geigy) and U.S. Pat. No. 5,614,395. A preferred promoter forchemical induction is the tobacco PR-1a promoter.

A preferred category of promoters is that which is wound inducible.Numerous promoters have been described which are expressed at woundsites and also at the sites of phytopathogen infection. Ideally, such apromoter should only be active locally at the sites of infection, and inthis way the insecticidal modified Cry3A toxins only accumulate in cellsthat need to synthesize the insecticidal modified Cry3A toxins to killthe invading insect pest. Preferred promoters of this kind include thosedescribed by Stanford et al. Mol. Gen. Genet. 215:200-208 (1989), Xu etal. Plant Molec. Biol. 22:573-588 (1993), Logemann et al. Plant Cell1:151-158 (1989), Rohrmeier & Lehle, Plant Molec. Biol. 22:783-792(1993), Firek et al. Plant Molec. Biol. 22:129-142 (1993), and Warner etal. Plant J. 3:191-201 (1993).

Tissue-specific or tissue-preferential promoters useful for theexpression of the modified Cry3A toxin genes in plants, particularlymaize, are those which direct expression in root, pith, leaf or pollen,particularly root. Such promoters, e.g. those isolated from PEPC ortrpA, are disclosed in U.S. Pat. No. 5,625,136, or MTL, disclosed inU.S. Pat. No. 5,466,785. Both U.S. patents are herein incorporated byreference in their entirety.

Further preferred embodiments are transgenic plants expressing thenucleotide sequences in a wound-inducible or pathogeninfection-inducible manner.

In addition to promoters, a variety of transcriptional terminators arealso available for use in chimeric gene construction using the modifiedCry3A toxin genes of the present invention. Transcriptional terminatorsare responsible for the termination of transcription beyond thetransgene and its correct polyadenylation. Appropriate transcriptionalterminators and those that are known to function in plants include theCaMV 35S terminator, the tml terminator, the nopaline synthaseterminator, the pea rbcS E9 terminator and others known in the art.These can be used in both monocotyledons and dicotyledons. Any availableterminator known to function in plants can be used in the context ofthis invention.

Numerous other sequences can be incorporated into expression cassettesdescribed in this invention. These include sequences that have beenshown to enhance expression such as intron sequences (e.g. from Adhl andbronzel) and viral leader sequences (e.g. from TMV, MCMV and AMY).

It may be preferable to target expression of the nucleotide sequences ofthe present invention to different cellular localizations in the plant.In some cases, localization in the cytosol may be desirable, whereas inother cases, localization in some subcellular organelle may bepreferred. Subcellular localization of transgene-encoded enzymes isundertaken using techniques well known in the art. Typically, the DNAencoding the target peptide from a known organelle-targeted gene productis manipulated and fused upstream of the nucleotide sequence. Many suchtarget sequences are known for the chloroplast and their functioning inheterologous constructions has been shown. The expression of thenucleotide sequences of the present invention is also targeted to theendoplasmic reticulum or to the vacuoles of the host cells. Techniquesto achieve this are well known in the art.

Vectors suitable for plant transformation are described elsewhere inthis specification. For Agrobacterium-mediated transformation, binaryvectors or vectors carrying at least one T-DNA border sequence aresuitable, whereas for direct gene transfer any vector is suitable andlinear DNA containing only the construction of interest may bepreferred. In the case of direct gene transfer, transformation with asingle DNA species or co-transformation can be used (Schocher et al.Biotechnology 4:1093-1096 (1986)). For both direct gene transfer andAgrobacterium-mediated transfer, transformation is usually (but notnecessarily) undertaken with a selectable marker that may provideresistance to an antibiotic (kanamycin, hygromycin or methotrexate) or aherbicide (basta). Plant transformation vectors comprising the modifiedCry3A toxin genes of the present invention may also comprise genes (e.g.phosphomannose isomerase; PMI) which provide for positive selection ofthe transgenic plants as disclosed in U.S. Pat. Nos. 5,767,378 and5,994,629, herein incorporated by reference. The choice of selectablemarker is not, however, critical to the invention.

In another embodiment, a nucleotide sequence of the present invention isdirectly transformed into the plastid genome. A major advantage ofplastid transformation is that plastids are generally capable ofexpressing bacterial genes without substantial codon optimization, andplastids are capable of expressing multiple open reading frames undercontrol of a single promoter. Plastid transformation technology isextensively described in U.S. Pat. Nos. 5,451,513, 5,545,817, and5,545,818, in PCT application no. WO 95/16783, and in McBride et al.(1994) Proc. Natl. Acad. Sci. USA 91, 7301-7305. The basic technique forchloroplast transformation involves introducing regions of clonedplastid DNA flanking a selectable marker together with the gene ofinterest into a suitable target tissue, e.g., using biolistics orprotoplast transformation (e.g., calcium chloride or PEG mediatedtransformation). The 1 to 1.5 kb flanking regions, termed targetingsequences, facilitate homologous recombination with the plastid genomeand thus allow the replacement or modification of specific regions ofthe plastome. Initially, point mutations in the chloroplast 16S rRNA andrps12 genes conferring resistance to spectinomycin and/or streptomycinare utilized as selectable markers for transformation (Svab, Z.,Hajdukiewicz, P., and Maliga, P. (1990) Proc. Natl. Acad. Sci. USA 87,8526-8530; Staub, J. M., and Maliga, P. (1992) Plant Cell 4, 39-45).This resulted in stable homoplasmic transformants at a frequency ofapproximately one per 100 bombardments of target leaves. The presence ofcloning sites between these markers allowed creation of a plastidtargeting vector for introduction of foreign genes (Staub, J. M., andMaliga, P. (1993) EMBO J. 12, 601-606). Substantial increases intransformation frequency are obtained by replacement of the recessiverRNA or r-protein antibiotic resistance genes with a dominant selectablemarker, the bacterial aadA gene encoding the spectinomycin-cletoxifyingenzyme aminoglycoside-3′-adenyltransf erase (Svab, Z., and Maliga, P.(1993) Proc. Natl. Acad. Sci. USA 90, 913-917). Previously, this markerhad been used successfully for high-frequency transformation of theplastid genome of the green alga Chlamydomonas reinhardtii(Goldschmidt-Clermont, M. (1991) Nucl. Acids Res. 19:4083-4089). Otherselectable markers useful for plastid transformation are known in theart and encompassed within the scope of the invention. Typically,approximately 15-20 cell division cycles following transformation arerequired to reach a homoplastidic state. Plastid expression, in whichgenes are inserted by homologous recombination into all of the severalthousand copies of the circular plastid genome present in each plantcell, takes advantage of the enormous copy number advantage overnuclear-expressed genes to permit expression levels that can readilyexceed 10% of the total soluble plant protein. In a preferredembodiment, a nucleotide sequence of the present invention is insertedinto a plastid-targeting vector and transformed into the plastid genomeof a desired plant host. Plants homoplastic for plastid genomescontaining a nucleotide sequence of the present invention are obtained,and are preferentially capable of high expression of the nucleotidesequence.

Combinations of Insect Control Principles

The modified Cry3A toxins of the invention can be used in combinationwith Bt δ-endotoxins or other pesticidal principles to increase pesttarget range. Furthermore, the use of the modified Cry3A toxins of theinvention in combination with Bt δ-endotoxins or other pesticidalprinciples of a distinct nature has particular utility for theprevention and/or management of insect resistance.

Other insecticidal principles include, for example, lectins, α-amylase,peroxidase and cholesterol oxidase. Vegetative Insecticidal Proteingenes, such as vip1A(a) and vip2A(a) as disclosed in U.S. Pat. No.5,889,174 and herein incorporated by reference, are also useful in thepresent invention.

This co-expression of more than one insecticidal principle in the sametransgenic plant can be achieved by genetically engineering a plant tocontain and express all the genes necessary. Alternatively, a plant,Parent 1, can be genetically engineered for the expression of genes ofthe present invention. A second plant, Parent 2, can be geneticallyengineered for the expression of a supplemental insect controlprinciple. By crossing Parent 1 with Parent 2, progeny plants areobtained which express all the genes introduced into Parents 1 and 2.

Transgenic seed of the present invention can also be treated with aninsecticidal seed coating as described in U.S. Pat. Nos. 5,849,320 and5,876,739, herein incorporated by reference. Where both the insecticidalseed coating and the transgenic seed of the invention are active againstthe same target insect, the combination is useful (i) in a method forenhancing activity of a modified Cry3A toxin of the invention againstthe target insect and (ii) in a method for preventing development ofresistance to a modified Cry3A toxin of the invention by providing asecond mechanism of action against the target insect. Thus, theinvention provides a method of enhancing activity against or preventingdevelopment of resistance in a target insect, for example corn rootworm,comprising applying an insecticidal seed coating to a transgenic seedcomprising one or more modified Cry3A toxins of the invention.

Even where the insecticidal seed coating is active against a differentinsect, the insecticidal seed coating is useful to expand the range ofinsect control, for example by adding an insecticidal seed coating thathas activity against lepidopteran insects to the transgenic seed of theinvention, which has activity against coleopteran insects, the coatedtransgenic seed produced controls both lepidopteran and coleopteraninsect pests.

EXAMPLES

The invention will be further described by reference to the followingdetailed examples. These examples are provided for the purposes ofillustration only, and are not intended to be limiting unless otherwisespecified. Standard recombinant DNA and molecular cloning techniquesused here are well known in the art and are described by J. Sambrook, etal., Molecular Cloning: A Laboratory Manual. 3d Ed., Cold Spring Harbor,N.Y.: Cold Spring Harbor Laboratory Press (2001); by T. J. Silhavy, M.L. Berman, and L. W. Enquist, Experiments with Gene Fusions, Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y. (1984) and by Ausubel, F. M,et al., Current Protocols in Molecular Biology, New York, John Wiley andSons Inc., (1988), Reiter, et al., Methods in Arabidopsis Research.World Scientific Press (1992), and Schultz et al., Plant MolecularBiology Manual, Kluwer Academic Publishers (1998).

Example 1 Maize Optimized cry3A Gene Construction

The maize optimized cry3A gene was made according to the proceduredisclosed in U.S. Pat. No. 5,625,136, herein incorporated by referencein its entirety. In this procedure, maize preferred codons, i.e., thesingle codon that most frequently encodes that amino acid in maize, isused. The maize preferred codon for a particular amino acid is derivedfrom known gene sequences from maize. Maize codon usage for 28 genesfrom maize plants is found in Murray et al., Nucleic Acids Research17:477-498 (1989). The synthetic cry3A sequence made with maizeoptimized codons is set forth in SEQ ID NO: 3.

Example 2 Identification of Cathepsin-G Enzymatic Activity in WesternCorn Rootworm Guts

Cathepsin G-like (serine protease) and cathepsin B-like (cysteineprotease) enzymatic activities in western corn rootworm guts weremeasured using colorimetric substrates. Each 1 ml reaction containedabout five homogenized midguts of the 3rd instar of western cornrootworm and approximately 1 mg of substrate dissolved in reactionbuffer (10 mM Tris, 5 mM NaCl, 0.01 M DTT, pH 7.5). The cathepsin Gsubstrate tested was Ala-Ala-Pro-Phe (SEQ ID NO: 35)-pNA and cathepsin Bsubstrate, Arg-Arg-pNA. The reactions were incubated at approximately28° C. for 1 hr. The intensity of yellow color formation, indicative ofthe efficiency of a protease to recognize the appropriate substrate, wascompared in treatments vs. controls. The reactions were scored asnegative (−) if no color or slight background color was detected.Reactions which were 25%, 50%, 75% or 100% above background were scoredas +, ++, +++, or ++++, respectively. Results of the enzymatic assaysare shown in the table 1.

TABLE 1 Results of Western Corn Rootworm Gut Protease Assay. ReactionProduct Color intensity WCR gut only − Cathepsin B substrate only −Cathepsin G substrate only − WCR gut + Cathepsin B substrate + WCR gut +Cathepsin G substrate +++

This is the first time that the serine protease cathepsin G activity hasbeen identified in western corn rootworm guts. Western corn rootwormguts clearly have stronger cathepsin G, the serine protease, activitycompared to cathepsin B, the cysteine protease, activity. The AAPFsequence (SEQ ID NO: 35) was selected as the cathepsin G proteaserecognition site for creating modified Cry3A toxins of the presentinvention.

Example 3 Construction of Modified cry3A Genes

Modified cry3A genes comprising a nucleotide sequence that encodes thecathepsin G recognition site in domain I, domain III, or domain I anddomain III were made using overlap PCR. The maize optimized cry3A gene(SEQ ID NO: 2), comprised in plasmid pCIB6850 (SEQ ID NO: 5), was usedas the starting template. Eight modified cry3A gene constructs, whichencode modified Cry3A toxins, were made; cry3A054, cry3A055, andcry3A085, which comprise the cathepsin G recognition site codingsequence in domain I; cry3A058, cry3A082, which comprise the cathepsin Grecognition site coding sequence in domain III; cry3A056, cry3A057,cry13A083, which comprise the cathepsin G recognition site codingsequence in domain I and domain III. The eight modified cry3A genes andthe modified Cry3A toxins they encode are described as follows:

cry3A054 Comprised in pCMS054

cry3A054 (SEQ ID NO: 6) comprises a nucleotide sequence encoding amodified Cry3A toxin. Three overlap PCR primer pairs were used to insertthe nucleotide sequence encoding the cathepsin G recognition site intothe maize optimized cry3A gene:

1. BamExt1-5′-GGATCCACCATGACGGCCGAC-3′ (SEQ ID NO: 22)AAPFtail3-5′-GAACGGTGCAGCGGGGTTCTTCTGCCAGC-3′ (SEQ ID NO: 23) 2.Tail5mod-5′-GCTGCACCGTTCCCCCACAGCCAGGGCCG-3′ (SEQ ID NO: 24)XbaIExt2-5′-TCTAGACCCACGTTGTACCAC-3′ (SEQ ID NO: 25) 3.BamExt1-5′-GGATCCACCATGACGGCCGAC-3′ (SEQ ID NO: 22)XbaIExt2-5′-TCTAGACCCACGTTGTACCAC-3′ (SEQ ID NO: 25)

Primer pair 1 and primer pair 2 generated two unique PCR products. Theseproducts were then combined in equal parts and primer pair 3 was used tojoin the products to generate one PCR fragment that was cloned back intothe original pCIB6850 template. The modified cry3A054 gene was thentransferred to pBluescript (Stratagene). The resulting plasmid wasdesignated pCMS054 and comprises the cry3A054 gene (SEQ ID NO: 6).

The modified Cry3A054 toxin (SEQ ID NO: 7), encoded by the modifiedcry3A gene comprised in pCMS054, has a cathepsin G recognition site,comprising the amino acid sequence AAPF (SEQ ID NO: 35), inserted indomain I between amino acids 107 and 113 of the Cry3A toxin set forth inSEQ ID NO: 4. The cathepsin G recognition site replaces the naturallyoccurring trypsin recognition site and is adjacent to a naturallyoccurring chymotrypsin recognition site.

cry3A055 Comprised in pCMS055

cry3A055 (SEQ ID NO: 8) comprises a nucleotide sequence encoding amodified Cry3A toxin. Three overlap PCR primer pairs were used to insertthe nucleotide sequence encoding the cathepsin G recognition site intothe maize optimized cry3A gene:

1. BamExt1-5′-GGATCCACCATGACGGCCGAC-3′ (SEQ ID NO: 22)AAPFtail3-5′-GAACGGTGCAGCGGGGTTCTTCTGCCAGC-3′ (SEQ ID NO: 23) 2.AAPFtail4-5′-GCTGCACCGTTCCGCAACCCCCACAGCCA-3′ (SEQ ID NO: 26)XbaIExt2-5′-TCTAGACCCACGTTGTACCAC-3′ (SEQ ID NO: 25) 3.BamExt1-5′-GGATCCACCATGACGGCCGAC-3′ (SEQ ID NO: 22)XbaIExt2-5′-TCTAGACCCACGTTGTACCAC-3′ (SEQ ID NO: 25)

Primer pair 1 and primer pair 2 generated two unique PCR products. Theseproducts were then combined in equal parts and primer pair 3 was used tojoin the products to generate one PCR fragment that was cloned back intothe original pCIB6850 template. The modified cry3A055 gene was thentransferred to pBluescript (Stratagene). The resulting plasmid wasdesignated pCMS055 and comprises the cry3A055 gene (SEQ ID NO: 8).

The modified Cry3A055 toxin (SEQ ID NO: 9), encoded by the modifiedcry3A gene comprised in pCMS055, has a cathepsin G recognition sitecomprising the amino acid sequence AAPF (SEQ ID NO: 35) inserted indomain I between amino acids 107 and 111 of the Cry3A toxin set forth inSEQ ID NO: 4. The cathepsin G recognition site is adjacent to a naturaltrypsin and chymotrypsin recognition site.

cry3A058 Comprised in pCMS058

cry3A058 (SEQ ID NO: 14) comprises a nucleotide sequence encoding amodified Cry3A toxin. Three overlap PCR primer pairs were used to insertthe nucleotide sequence encoding the cathepsin G recognition site intothe maize optimized cry3A gene:

1. SalExt-5′-GAGCGTCGACTTCTTCAAC-3′ (SEQ ID NO: 27)AAPF-Y2-5′-GAACGGTGCAGCGTATTGGTTGAAGGGGGC-3′ (SEQ ID NO: 28) 2.AAPF-Y1-5′-GCTGCACCGTTCTACTTCGACAAGACCATC-3′ (SEQ ID NO: 29)SacExt-5′-GAGCTCAGATCTAGTTCACGG-3′ (SEQ ID NO: 30) 3.SalExt-5′-GAGCGTCGACTTCTTCAAC-3′ (SEQ ID NO: 27)SacExt-5′-GAGCTCAGATCTAGTTCACGG-3′ (SEQ ID NO: 30)

Primer pair 1 and primer pair 2 generated two unique PCR products. Theseproducts were then combined in equal parts and primer pair 3 was used tojoin the products to generate one PCR fragment that was cloned back intothe original pCIB6850 template. The modified cry3A058 gene was thentransferred to pBluescript (Stratagene). The resulting plasmid wasdesignated pCMS058 and comprises the cry3A058 gene (SEQ ID NO: 14).

The modified Cry3A058 toxin (SEQ ID NO: 15), encoded by the modifiedcry3A gene, has a cathepsin G recognition site, comprising the aminoacid sequence AAPF (SEQ ID NO: 35), inserted in domain III between aminoacids 540 and 541 of the Cry3A toxin set forth in SEQ ID NO: 4. Thecathepsin G recognition site is within a naturally occurringchymotrypsin recognition site.

pCMS082 Comprising cry3A082

cry3A082 (SEQ ID NO: 12) comprises a nucleotide sequence encoding amodified Cry3A toxin. A QuikChange Site Directed Mutagenesis PCR primerpair was used to insert the nucleotide sequence encoding the cathepsin Grecognition site into the maize optimized cry3A gene:

(SEQ ID NO: 31) BBmod1-5′-CGGGGCCCCCGCTGCACCGTTCTACTTCGACA-3′(SEQ ID NO: 32) BBmod2-5′-TGTCGAAGTAGAACGGTGCAGCGGGGGCCCCG-3′

The primer pair generated a unique PCR product. This product was clonedback into the original pCIB6850 template. The modified cry3A082 gene wasthen transferred to pBluescript (Stratagene). The resulting plasmid wasdesignated pCMS082 and comprises the cry3A082 gene (SEQ ID NO: 12).

The modified Cry3A082 toxin (SEQ ID NO: 13), encoded by the modifiedcry3A gene, has a cathepsin G recognition site, comprising the aminoacid sequence AAPF (SEQ ID NO: 35), inserted in domain III between aminoacids 539 and 542 of the Cry3A toxin set forth in SEQ ID NO: 4. Thecathepsin G recognition site replaces a naturally occurring chymotrypsinrecognition site.

cry3A056 Comprised in pCMS056

cry3A056 (SEQ ID NO: 18) comprises a nucleotide sequence encoding amodified Cry3A toxin. Six overlap PCR primer pairs were used to inserttwo cathepsin G recognition sites into the maize optimized cry3A gene:

1. BamExt1-5′-GGATCCACCATGACGGCCGAC-3′ (SEQ ID NO: 22)AAPFtail3-5′-GAACGGTGCAGCGGGGTTCTTCTGCCAGC-3′ (SEQ ID NO: 23) 2.AAPFtail4-5'-GCTGCACCGTTCCGCAACCCCCACAGCCA-3′ (SEQ ID NO: 26)XbaIExt2-5′-TCTAGACCCACGTTGTACCAC-3′ (SEQ ID NO: 25) 3.BamExt1-5′-GGATCCACCATGACGGCCGAC-3′ (SEQ ID NO: 22)XbaIExt2-5′-TCTAGACCCACGTTGTACCAC-3′ (SEQ ID NO: 25) 4.SalExt-5′-GAGCGTCGACTTCTTCAAC-3′ (SEQ ID NO: 27)AAPF-Y2-5′-GAACGGTGCAGCGTATTGGTTGAAGGGGGC-3′ (SEQ ID NO: 28) 5.AAPF-Y1-5′-GCTGCACCGTTCTACTTCGACAAGACCATC-3′ (SEQ ID NO: 29)SacExt-5′-GAGCTCAGATCTAGTTCACGG-3′ (SEQ ID NO: 30) 6.SalExt-5′-GAGCGTCGACTTCTTCAAC-3′ (SEQ ID NO: 27)SacExt-5′-GAGCTCAGATCTAGTTCACGG-3′ (SEQ ID NO: 30)

Primer pair 1 and primer pair 2 generated two unique PCR products. Theseproducts were combined in equal parts and primer pair 3 is used to jointhe products to generate one PCR fragment that was cloned back into theoriginal pCIB6850 plasmid. The modified cry3A055 gene was thentransferred to pBluescript (Stratagene). The resulting plasmid wasdesignated pCMS055. Primer pair 4 and primer pair 5 generated anotherunique set of fragments that were joined by another PCR with primer pair6. This fragment was cloned into domain III of the modified cry3A055gene comprised in pCMS055. The resulting plasmid was designated pCMS056and comprises the cry3A056 gene (SEQ ID NO: 18).

The modified Cry3A056 toxin (SEQ ID NO: 19), encoded by the modifiedcry3A gene, has a cathepsin G recognition site, comprising the aminoacid sequence AAPF (SEQ ID NO: 35), inserted in domain I between aminoacids 107 and 111 and in domain III between amino acids 540 and 541 ofthe Cry3A toxin set forth in SEQ ID NO: 4. The cathepsin G recognitionsite is adjacent to a naturally occurring trypsin and chymotrypsinrecognition site in domain I and is within a naturally occurringchymotrypsin recognition site in domain III.

cry3A057 Comprised in pCMS057

cry3A057 (SEQ ID NO: 16) comprises a nucleotide sequence encoding amodified Cry3A toxin. Six overlap PCR primer pairs are used to inserttwo cathepsin G recognition sites into the maize optimized cry3A gene:

1. BamExt1-5′-GGATCCACCATGACGGCCGAC-3′ (SEQ ID NO: 22)AAPFtail3-5′-GAACGGTGCAGCGGGGTTCTTCTGCCAGC-3′ (SEQ ID NO: 23) 2.Tail5mod-5′-GCTGCACCGTTCCCCCACAGCCAGGGCCG-3′ (SEQ ID NO: 24)XbaIExt2-5′-TCTAGACCCACGTTGTACCAC-3′ (SEQ ID NO: 25) 3.BamExt1-5′-GGATCCACCATGACGGCCGAC-3′ (SEQ ID NO: 22)XbaIExt2-5′-TCTAGACCCACGTTGTACCAC-3′ (SEQ ID NO: 25) 4.SalExt-5′-GAGCGTCGACTTCTTCAAC-3′ (SEQ ID NO: 27)AAPF-Y2-5′-GAACGGTGCAGCGTATTGGTTGAAGGGGGC-3′ (SEQ ID NO: 28) 5.AAPF-Y1-5′-GCTGCACCGTTCTACTTCGACAAGACCATC-3′ (SEQ ID NO: 29)SacExt-5′-GAGCTCAGATCTAGTTCACGG-3′ (SEQ ID NO: 30) 6.SalExt-5′-GAGCGTCGACTTCTTCAAC-3′ (SEQ ID NO: 27)SacExt-5′-GAGCTCAGATCTAGTTCACGG-3′ (SEQ ID NO: 30)

Primer pair 1 and primer pair 2 generated two unique PCR products. Theseproducts were combined in equal parts and primer pair 3 was used to jointhe products to generate one PCR fragment that was cloned back into theoriginal pCIB6850 plasmid. The modified cry3A054 gene was thentransferred to pBluescript (Stratagene). The resulting plasmid wasdesignated pCMS054. Primer pair 4 and primer pair 5 generated anotherunique set of fragments that were joined by another PCR with primer pair6. This fragment was cloned into domain III of the modified cry3A054gene comprised in pCMS054. The resulting plasmid was designated pCMS057and comprises the cry3A057 gene (SEQ ID NO: 16).

The modified Cry3A057 toxin (SEQ ID NO: 17), encoded by the modifiedcry3A gene, has a cathepsin G recognition site, comprising the aminoacid sequence AAPF (SEQ ID NO: 35), inserted in domain I between aminoacids 107 and 113 and in domain III between amino acids 540 and 541 ofthe Cry3A toxin set forth in SEQ ID NO: 4. The cathepsin G recognitionsite replaces a naturally occurring trypsin recognition site and isadjacent to a naturally occurring chymotrypsin recognition site indomain I and is within a naturally occurring chymotrypsin recognitionsite in domain III.

cry3A083 Comprised in pCMS083

cry3A083 (SEQ ID NO: 20) comprises a nucleotide sequence encoding amodified Cry3A toxin. Three overlap PCR primer pairs and one QuikChangeSite Directed Mutagenesis PCR primer pair were used to insert twocathepsin G recognition sites into the maize optimized cry3A gene:

1. BamExt1-5′-GGATCCACCATGACGGCCGAC-3′ (SEQ ID NO: 22)AAPFtail3-5′-GAACGGTGCAGCGGGGTTCTTCTGCCAGC-3′ (SEQ ID NO: 23) 2.AAPFtail4-5′-GCTGCACCGTTCCGCAACCCCCACAGCCA-3′ (SEQ ID NO: 26)XbaIExt2-5′-TCTAGACCCACGTTGTACCAC-3′ (SEQ ID NO: 25) 3.BamExt1-5′-GGATCCACCATGACGGCCGAC-3′ (SEQ ID NO: 22)XbaIExt2-5′-TCTAGACCCACGTTGTACCAC-3′ (SEQ ID NO: 25)BBmod1-5′-CGGGGCCCCCGCTGCACCGTTCTACTTCGACA-3 (SEQ ID NO: 31)BBmod2-5′-TGTCGAAGTAGAACGGTGCAGCGGGGGCCCCG-3′ (SEQ ID NO: 32)

Primer pair 1 and primer pair 2 generated two unique PCR products. Theseproducts were combined in equal parts and primer pair 3 was used to jointhe products to generate one PCR fragment that was cloned back into theoriginal pCIB6850 plasmid. The modified cry3A055 gene was thentransferred to pBluescript (Stratagene). The resulting plasmid wasdesignated pCMS055. Primer pair 4 generated another unique fragment thatwas cloned into domain III of the modified cry3A comprised in pCMS055.The resulting plasmid was designated pCMS083 and comprises the cry3A083gene (SEQ ID NO: 20).

The modified Cry3A083 toxin (SEQ ID NO: 21), encoded by the modifiedcry3A gene, has a cathepsin G recognition site, comprising the aminoacid sequence AAPF (SEQ ID NO: 35), inserted in domain I between aminoacids 107 and 111 and between amino acids 539 and 542 of the Cry3A toxinset forth in SEQ ID NO: 4. The cathepsin G recognition site is adjacentto a naturally occurring trypsin and chymotrypsin recognition site indomain I and replaces a naturally occurring chymotrypsin recognitionsite in domain III.

cry3A085 Comprised in pCMS085

The cry3A085 gene (SEQ ID NO: 10) comprises a cathepsin G codingsequence at the same position as in the cry3A055 gene described above.The cry3A085 gene has an additional 24 nucleotides inserted at the 5′end which encode amino acids 41-47 of the deduced amino acid sequenceset forth in SEQ ID NO: 2 as well as an additional methionine. Theadditional nucleotides are inserted at the 5′ end of the cry3A055 geneusing the following PCR primer pair:

mo3Aext- (SEQ ID NO: 33)5′-GGATCCACCATGAACTACAAGGAGTTCCTCCGCATGACCGCCGACA AC-3′ (SEQ ID NO: 34)CMS16-5′-CCTCCACCTGCTCCATGAAG-3′

The modified Cry3A085 toxin (SEQ ID NO: 11), encoded by the modifiedcry3A gene, has a cathepsin G recognition site, comprising the aminoacid sequence AAPF (SEQ ID NO: 35), inserted in domain I between aminoacids corresponding to 107 and 111 of the Cry3A toxin set forth in SEQID NO: 4 and has an additional eight amino acid residues at theN-terminus of which the second residue corresponds to amino acid number41 of the amino acid sequence set forth in SEQ ID NO: 2.

Example 4 Insecticidal Activity of Modified Cry3A Toxins

Modified Cry3A toxins were tested for insecticidal activity againstwestern corn rootworm, northern corn rootworm and southern corn rootwormin insect bioassays. Bioassays were performed using a diet incorporationmethod. E. coli clones that express one of the modified Cry3A toxins ofthe invention were grown overnight. 500 μl of an overnight culture wassonicated and then mixed with 500 μl of molten artificial diet (Marroneet al. (1985) J. of Economic Entomology 78:290-293). The molten diet wasdispensed into small petri dishes, allowed to solidify and then 20neonate corn rootworm were placed on the diet. The petri dishes wereheld at approximately 30° C. Mortality was recorded after approximately6 days. All of the modified Cry3A toxins cause 50%-100% mortality towestern and northern corn rootworm whereas the unmodified Cry3A toxincauses 0%-30% mortality. The modified Cry3A toxins had no activityagainst southern corn rootworm.

Example 5 Creation of Transgenic Maize Plants Comprising Modified cry3ACoding Sequences

Three modified cry3A genes, cry3A055, representative of a domainmodification, cry3A058, representative of a domain III modification, andcry3A056, representative of a domain I and domain III modification, werechosen for transformation into maize plants. An expression cassettecomprising a modified cry3A coding sequence is transferred to a suitablevector for Agrobacterium-mediated maize transformation. For thisexample, an expression cassette comprises, in addition to the modifiedcry3A gene, the MTL promoter (U.S. Pat. No. 5,466,785) and the nosterminator which is known in the art.

Transformation of immature maize embryos is performed essentially asdescribed in Negrotto et al., 2000, Plant Cell Reports 19: 798-803. Forthis example, all media constituents are as described in Negrotto etal., supra. However, various media constituents known in the art may besubstituted.

The genes used for transformation are cloned into a vector suitable formaize transformation. Vectors used in this example contain thephosphomannose isomerase (PMI) gene for selection of transgenic lines(Negrotto et al. (2000) Plant Cell Reports 19: 798-803).

Agrobacterium strain LBA4404 (pSB1) containing the plant transformationplasmid is grown on YEP (yeast extract (5 g/L), peptone (10 g/L), NaCl(5 g/L), 15g/l agar, pH 6.8) solid medium for 2-4 days at 28° C.Approximately 0.8×10⁹ Agrobacterium are suspended in LS-inf mediasupplemented with 100 μM As (Negrotto et al., (2000) Plant Cell Rep 19:798-803). Bacteria are pre-induced in this medium for 30-60 minutes.

Immature embryos from A188 or other suitable genotype are excised from8-12 day old ears into liquid LS-inf +100 μM As. Embryos are rinsed oncewith fresh infection medium. Agrobacterium solution is then added andembryos are vortexed for 30 seconds and allowed to settle with thebacteria for 5 minutes. The embryos are then transferred scutellum sideup to LSAs medium and cultured in the dark for two to three days.Subsequently, between 20 and 25 embryos per petri plate are transferredto LSDc medium supplemented with cefotaxime (250 mg/l) and silvernitrate (1.6 mg/l) and cultured in the dark for 28° C. for 10 days.

Immature embryos, producing embryogenic callus are transferred toLSD1M0.5S medium. The cultures are selected on this medium for 6 weekswith a subculture step at 3 weeks. Surviving calli are transferred toReg1 medium supplemented with mannose. Following culturing in the light(16 hour light/8 hour dark regiment), green tissues are then transferredto Reg2 medium without growth regulators and incubated for 1-2 weeks.Plantlets are transferred to Magenta GA-7 boxes (Magenta Corp, ChicagoIll.) containing Reg3 medium and grown in the light. After 2-3 weeks,plants are tested for the presence of the PMI genes and the modifiedcry3A genes by PCR. Positive plants from the PCR assay are transferredto the greenhouse and tested for resistance to corn rootworm.

Example 6 Analysis of Transgenic Maize Plants Corn Rootworm EfficacyRoot Excision Bioassay

Plants are sampled as they are being transplanted from Magenta GA-7boxes into soil. This allows the roots to be sampled from a reasonablysterile environment relative to soil conditions. Sampling consists ofcutting a small piece of root (ca. 2-4 cm long) and placing it ontoenriched phytagar (phytagar, 12 g., sucrose, 9 g., MS salts, 3 ml., MSvitamins, 3 ml., Nystatin (25 mg/ml), 3 ml., Cefotaxime (50 mg/ml), 7ml., Aureomycin (50 mg/ml), 7 ml., Streptomycin (50 mg/ml), 7 ml., dH2O,600 ml) in a small petri-dish. Negative controls are either transgenicplants that are PCR negative for the modified cry3A gene from the sameexperiment, or from non-transgenic plants (of a similar size to testplants) that are being grown in the phytotron. If sampling control rootsfrom soil, the root samples are washed with water to remove soilresidue, dipped in Nystatin solution (5 mg/ml), removed from the dip,blotted dry with paper toweling, and placed into a phytagar dish.

Root samples are inoculated with western corn rootworms by placing 10first instar larvae onto the inside surface of the lid of each phytagardish and the lids then tightly resealed. Larvae are handled using a finetip paintbrush. After all dishes are inoculated, the tray of dishes isplaced in the dark at room temperature until data collection.

At 3-4 days post inoculation, data is collected. The percent mortalityof the larvae is calculated along with a visual damage rating of theroot. Feeding damage is rated as high, moderate, low, or absent andgiven a numerical value of 3, 2, 1 or 0, respectively. Root samplescausing at least 40% mortality and having a damage rating of 2 or lessare considered positive.

Results in the following table show that plants expressing a modifiedCry3A toxin cause from 40-100% mortality to western corn rootwormwhereas control plants cause 0-30% mortality. Also, plants expressing amodified Cry3A toxin sustain significantly less feeding damage thancontrol plants.

TABLE 2 Results of Root Excision Bioassay. Percent Mortality T0 ModifiedCry3A Per Plant Mean Damage Event Toxin Expressed A B C D E Rating PerEvent 240A7 Cry3A055 80 40 80 60 0.8 240B2 Cry3A055 60 60 60 80 1.25240B9 Cry3A055 40 60 60 100 1 240B10 Cry3A055 80 40 60 60 1 240A15Cry3A055 80 60 50 70 70 0.6 240A5 Cry3A055 60 80 60 0.33 240A9 Cry3A05550 60 60 70 70 1.6 244A4 Cry3A058 50 1 244A7 Cry3A058 40 40 60 1.3 244A5Cry3A058 50 1 244B7 Cry3A058 90 1 244B6 Cry3A058 50 40 60 1 243A3Cry3A056 50 90 80 60 1.25 243A4 Cry3A056 50 80 60 1.7 243B1 Cry3A056 8090 0.5 243B4 Cry3A056 70 60 50 80 1.5 245B2 Cry3A056 90 50 70 60 1 WT1 —0 10 20 10 0 2.6 WT2 — 0 30 0 0 20 2.8

Whole Plant Bioassay

Some positive plants identified using the root excision bioassaydescribed above are evaluated for western corn rootworm resistance usinga whole plant bioassay. Plants are infested generally within 3 daysafter the root excision assay is completed.

Western corn rootworm eggs are preincubated so that hatch occurs 2-3days after plant inoculation. Eggs are suspended in 0.2% agar andapplied to the soil around test plants at approximately 200 eggs/plant.

Two weeks after the eggs hatch, plants are evaluated for damage causedby western corn rootworm larvae. Plant height attained, lodging, androot mass are criteria used to determine if plants are resistant towestern corn rootworm feeding damage. At the time of evaluation, controlplants typically are smaller than modified Cry3A plants. Also,non-transgenic control plants and plants expressing the unmodified Cry3Atoxin encoded by the maize optimized cry3A gene have lodged (stems loosein soil or stems at an angle of greater than 30° from vertical,including laying completely horizontal) during this time due to severepruning (completely cut) of most of the roots resulting in no root massaccumulation. At the time of evaluation, plants expressing a modifiedCry3A toxin of the invention are taller than control plants, have notlodged (stems remain tightly intact to soil and are vertical), and havea large intact root mass due to the insecticidal activity of themodified Cry3A toxin.

ELISA Assay

ELISA analysis according to the method disclosed in U.S. Pat. No.5,625,136 is used for the quantitative determination of the level ofmodified and unmodified Cry3A protein in transgenic plants.

TABLE 3 Whole Plant Bioassay Results and Protein Levels Transgenic Cry3AProtein Intact Maize Type of Cry3A Level in Plant Root Plant ToxinExpressed Roots (ng/mg) Lodged^(a) Mass^(b) 240A2E modified Cry3A055 224− + 240A9C modified Cry3A055 71 − + 240B9D modified Cry3A055 204 − +240B9E modified Cry3A055 186 − + 240B10D modified Cry3A055 104 − +240B10E modified Cry3A055 70 − + 240A15E modified Cry3A055 122 − +240B4D modified Cry3A055 97 − + 243B5A modified Cry3A056 41 − + 244A7Amodified Cry3A058 191 − + 710-2-51 maize optimized 39 + − 710-2-54 maizeoptimized 857 + − 710-2-61 maize optimized 241 + − 710-2-67 maizeoptimized 1169 + − 710-2-68 maize optimized 531 + − 710-2-79 maizeoptimized 497 + − 710-2-79 maize optimized 268 + − WT1 Control — 0 + −WT2 Control — 0 + − ^(a)A “−” indicates that the plant was standingvertical and therefore not lodged; a “+” indicates that the plant stemwas greater than 30° from vertical, including laying completelyhorizontal. ^(b)A “+” indicates that the plant had an intact root massthat was not pruned by corn rootworm feeding; a “−” indicates that theplant had little or no intact root mass due to severe pruning of theroots.

1. A modified Cry3A toxin comprising a non-naturally occurring proteaserecognition site, wherein said protease recognition site modifies aCry3A toxin and is located at a position selected from the groupconsisting of: a. between amino acids corresponding to amino acidnumbers 536 and 542 of SEQ ID NO: 4; and b. between amino acidscorresponding to amino acid numbers 107 and 111 of SEQ ID NO: 4, andbetween amino acids corresponding to amino acid numbers 536 and 542 ofSEQ ID NO: 4, wherein said protease recognition site is recognizable bya gut protease of western corn rootworm, and wherein said modified Cry3Atoxin causes higher mortality to western corn rootworm than themortality caused by said Cry3A toxin to western corn rootworm in anartificial diet bioassay.
 2. The modified Cry3A toxin according to claim1, wherein said gut protease is a serine protease or a cysteineprotease.
 3. The modified Cry3A toxin according to claim 1, wherein saidprotease recognition site is located at a position selected from: a.between amino acid numbers 536 and 542 of SEQ ID NO: 4, and b. betweenamino acid numbers 107 and 111 of SEQ ID NO: 4, and between amino acidnumbers 536 and 542 of SEQ ID NO:
 4. 4. The modified Cry3A toxinaccording to claim 1 which is active against northern corn rootworm. 5.A composition comprising an effective amount of the modified Cry3A toxinof claim 1 to cause mortality to western com rootworm.
 6. An isolatednucleic acid molecule comprising a nucleotide sequence that encodes amodified Cry3A toxin comprising a non-naturally occurring proteaserecognition site, wherein said protease recognition site modifies aCry3A toxin and is located at a position selected from the groupconsisting of: a. between amino acids corresponding to amino acidnumbers 536 and 542 of SEQ ID NO: 4; and b. between amino acidscorresponding to amino acid numbers 107 and 111 of SEQ ID NO: 4, andbetween amino acids corresponding to amino acid numbers 536 and 542 ofSEQ ID NO: 4, wherein said protease recognition site is recognizable bya gut protease of western corn rootworm, and wherein said modified Cry3Atoxin causes higher mortality to western corn rootworm than themortality caused by said Cry3A toxin to western corn rootworm in anartificial diet bioassay.
 7. The isolated nucleic acid moleculeaccording to claim 6, wherein said gut protease is a serine protease ora cysteine protease.
 8. The isolated nucleic acid molecule according toclaim 6, wherein said protease recognition site is located at a positionselected from: a, between amino acid numbers 536 and 542 of SEQ ID NO:4, and b. between amino acid numbers 107 and 111 of SEQ ID NO: 4, andbetween amino acid numbers 536 and 542 of SEQ ID NO:
 4. 9. The isolatednucleic acid molecule according to claim 6, wherein said modified Cry3Atoxin is active against northern corn rootworm.
 10. A chimeric constructcomprising a heterologous promoter sequence operatively linked to thenucleic acid molecule of claim
 6. 11. A recombinant vector comprisingthe chimeric construct of claim
 10. 12. A transgenic non-human host cellcomprising the chimeric construct of claim
 10. 13. The transgenicnon-human host cell according to claim 12, which is a plant cell.
 14. Anisolated toxin produced by the expression or the nucleic acid moleculeaccording to claim
 1. 15. A method of producing a modified Cry3A,comprising: a. obtaining the transgenic host cell according to claim 7;b. culturing the transgenic host cell under conditions that allow theexpression of the modified Cry3A toxin; and c. recovering the expressedmodified Cry3A toxin.
 16. A method of producing insect-resistant plants,comprising: a. stably integrating the nucleic acid molecule according toclaim 1 into the genome of plant cells; and b. regenerating stablytransformed plants from said transformed plant cells, wherein saidstably transformed plants express an effective amount of a modifiedCry3A toxin to render said transformed plant resistant to at leastwestern corn rootworm.
 17. A method of making a modified Cry3A toxin,comprising: a. obtaining a cry3A gene which encodes a Cry3A toxin; b.obtaining a nucleotide sequence which encodes a protease recognitionsite; c. inserting said nucleotide sequence into said cry3A gene, suchthat said protease recognition site is located in said Cry3A toxin at aposition between amino acids corresponding to amino acid numbers 107 and111 of SEQ ID NO: 4, or between amino acids corresponding to amino acidnumbers 107 and 111 of SEQ ID NO: 4 and between amino acidscorresponding to amino acid numbers 536 and 542 of SEQ ID NO: 4, thuscreating a modified cry3A gene; d. inserting said modified cry3A geneinto an expression cassette; and e. transforming said expressioncassette into a non-human host cell, wherein said host cell produces amodified Cry3A toxin.